Use of cyclic dipeptides in skin-aging slowing products

Abstract

The present invention relates to the field of microbiology and to the field of cosmetics and pharmaceuticals, in particular to a fungal fermentation product comprising a cyclic dipeptide, to a method for its preparation and to its use in a product for slowing down skin aging, and to the use of a cyclic dipeptide in a product for slowing down skin aging.

Description

Technical Field

[0001] This invention relates to the fields of microbiology, cosmetics, and pharmaceuticals, specifically to fungal fermentation products containing cyclic dipeptides, their preparation methods, and their application in products that slow down skin aging, and further to the application of cyclic dipeptides in products that slow down skin aging. Background Technology

[0002] With the improvement of living standards, people's pursuit of quality of life and their attention to health and beauty are increasing day by day. Skin, as the largest organ of the human body, not only protects the body from external harm but is also an important reflection of personal charm and health. As we age, skin gradually shows signs of aging, such as wrinkles, sagging, and age spots—all part of the natural physiological process. People are exploring ways to more effectively combat skin aging. This trend reflects society's emphasis on health and beauty and embodies people's relentless pursuit of a higher quality of life.

[0003] For example, patent application CN110731928A discloses a concentrated birch sap with significant anti-aging effects, capable of promoting collagen synthesis in the extracellular matrix, reducing the expression of matrix metalloproteinases, promoting hyaluronic acid production, and strengthening the structural integrity and functionality of the epidermis, dermis, and dermal-epidermal junction. For example, patent application CN201410755706.9 discloses a short peptide composed of 13 amino acids, which can stimulate cells to secrete large amounts of collagen, exhibiting significant anti-aging effects and potentially finding wide application in the biopharmaceutical and cosmetic industries.

[0004] Medicinal fungi are a class of microorganisms that, during their growth and development, produce bioactive components within their mycelium, sclerotia, or fruiting bodies. These bioactive components include polysaccharides, peptides, triterpenoids, alkaloids, and phenolic compounds, exhibiting a variety of biological activities such as antitumor, antiviral, anti-inflammatory, and antioxidant effects. Utilizing deep culture techniques for medicinal fungi can significantly shorten the preparation cycle of active ingredients. Summary of the Invention

[0005] The inventors unexpectedly discovered that cyclic dipeptide compounds contained in products prepared by fermentation of medicinal fungi have a significant effect in slowing down skin aging. Through research, the inventors found that cyclic dipeptide compounds can significantly reduce the activity of the aging marker SA-gal in a fibroblast aging model; significantly reduce tail fin wrinkling in a zebrafish photoaging model; and human trials show that cyclic dipeptides have effects such as reducing facial wrinkles. Therefore, this application provides the following invention:

[0006] In a first aspect, this application provides a fungal fermentation product comprising one or more of the following cyclic dipeptides or their salts: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0007] Cyclic dipeptides belong to the 2,5-dikepiperazine class of compounds (DKPs), which have a six-membered ring structure formed by the head-to-tail condensation of two amino acids through peptide bonds. In this document, the order of amino acids described in cyclic dipeptides is not restricted. For example, cyclic isoleucine-leucine Cyclo(ile-leu) and cyclic leucine-isoleucine Cyclo(leu-ile) represent the same cyclic dipeptide.

[0008] The inventors discovered that the fermentation products of some medicinal fungi contain cyclic dipeptides as described above, which have a significant effect on slowing down skin aging.

[0009] In some embodiments, the fungal fermentation product is a fungal fermentation filtrate containing one or more cyclic dipeptides or their salts in the following amounts: 0.15 ng / mL to 1.8 ng / mL cyclic proline-tyrosine, 2.0 ng / mL to 6.1 ng / mL cyclic isoleucine-proline, 2.5 ng / mL to 48.7 ng / mL cyclic proline-leucine, 2.0 ng / mL to 8.0 ng / mL cyclic proline-phenylalanine, 0.06 ng / mL to 0.39 ng / mL cyclic valine-phenylalanine, and 0.05 ng / mL to 0.92 ng / mL cyclic leucine-leucine.

[0010] In some embodiments, the fungal fermentation filtrate contains cycloproline-tyrosine or its salts in the following amounts: 0.150 ng / mL to 0.154 ng / mL, 0.154 ng / mL to 0.2 ng / mL, 0.2 ng / mL to 0.244 ng / mL, 0.244 ng / mL to 0.250 ng / mL, 0.25 ng / mL to 0.3 ng / mL, 0.3 ng / mL to 0.5 ng / mL, 0.5 ng / mL to 0.7 ng / mL, 0.7 ng / mL to 0.9 ng / mL, 0.9 ng / mL to 1.1 ng / mL, 1.1 ng / mL to 1.3 ng / mL, 1.3 ng / mL to 1.5 ng / mL, 1.5 ng / mL to 1.7 ng / mL, 1.7 ng / mL to 1.789 ng / mL, or 1.789 ng / mL to 1.180 ng / mL.

[0011] In some embodiments, the fungal fermentation filtrate contains cyclic isoleucine-proline or its salts in the following amounts: 2.0 ng / mL to 2.045 ng / mL, 2.045 ng / mL to 2.10 ng / mL, 2.1 ng / mL to 2.5 ng / mL, 2.5 ng / mL to 3.0 ng / mL, 3.0 ng / mL to 3.5 ng / mL, 3.5 ng / mL to 4.0 ng / mL, 4.0 ng / mL to 4.0 ng / mL. .5ng / mL, 4.5ng / mL~5.0ng / mL, 5.0ng / mL~5.1ng / mL, 5.1ng / mL~5.126ng / mL, 5.126ng / mL~5.20n g / mL, 5.2ng / mL~5.5ng / mL, 5.5ng / mL~6.0ng / mL, 6.0ng / mL~6.08ng / mL or 6.08ng / mL~6.10ng / mL.

[0012] In some embodiments, the fungal fermentation filtrate contains cyclic proline-leucine or its salts in the following amounts: 2.50 ng / mL to 2.585 ng / mL, 2.585 ng / mL to 2.60 ng / mL, 2.6 ng / mL to 3 ng / mL, 3 ng / mL to 5 ng / mL, 5 ng / mL to 7 ng / mL, 7 ng / mL to 9 ng / mL, 9.0 ng / mL to 9.469 ng / mL, 9.469 ng / mL. g / mL~9.50ng / mL, 9.5ng / mL~10ng / mL, 10ng / mL~20ng / mL, 20ng / mL~30ng / mL, 30ng / mL~40ng / mL, 4 0ng / mL~48ng / mL, 48.0ng / mL~48.6ng / mL, 48.60ng / mL~48.664ng / mL or 48.664ng / mL~48.70ng / mL.

[0013] In some embodiments, the fungal fermentation filtrate contains cycloproline-phenylalanine or its salts in the following amounts: 2.0 ng / mL to 2.082 ng / mL, 2.082 ng / mL to 2.10 ng / mL, 2.1 ng / mL to 2.3 ng / mL, 2.3 ng / mL to 2.5 ng / mL, 2.5 ng / mL to 2.7 ng / mL, 2.70 ng / mL to 2.78 ng / mL, 2.78 ng / mL to 2.80 ng / mL, 2.8 ng / mL to 3 ng / mL, 3 ng / mL to 4 ng / mL, 4 ng / mL to 5 ng / mL, 5 ng / mL to 6 ng / mL, 6 ng / mL to 7 ng / mL, 7.0 ng / mL to 7.5 ng / mL, 7.5 ng / mL to 7.9 ng / mL, 7.90 ng / mL to 7.94 ng / mL, or 7.94 ng / mL to 8.0 ng / mL.

[0014] In some embodiments, the fungal fermentation filtrate contains cyclovaline-phenylalanine or its salts in the following amounts: 0.06 ng / mL to 0.062 ng / mL, 0.062 ng / mL to 0.065 ng / mL, 0.065 ng / mL to 0.07 ng / mL, 0.07 ng / mL to 0.1 ng / mL, 0.1 ng / mL to 0.2 ng / mL, 0.20 ng / mL to 0.25 ng / mL, 0.25 ng / mL to 0.29 ng / mL, 0.290 ng / mL to 0.298 ng / mL, 0.298 ng / mL to 0.30 ng / mL, 0.30 ng / mL to 0.35 ng / mL, 0.35 ng / mL to 0.38 ng / mL, 0.380 ng / mL to 0.387 ng / mL, or 0.387 ng / mL to 0.39 ng / mL.

[0015] In some embodiments, the fungal fermentation filtrate contains cycloleucine-leucine or its salts in the following amounts: 0.050 ng / mL to 0.055 ng / mL, 0.055 ng / mL to 0.059 ng / mL, 0.059 ng / mL to 0.060 ng / mL, 0.06 ng / mL to 0.07 ng / mL, 0.07 ng / mL to 0.10 ng / mL, 0.10 ng / mL to 0.20 ng / mL, 0.20 ng / mL to 0.22 ng / mL, 0.220 ng / mL to 0.224 ng / mL, 0.224 ng / mL, etc. ng / mL~0.30ng / mL, 0.3ng / mL~0.4ng / mL, 0.4ng / mL~0.5ng / mL, 0.5ng / mL~0.6ng / mL, 0.6ng / mL~0.7ng / mL, 0.7ng / mL~0.8ng / mL , 0.8ng / mL~0.9ng / mL, 0.90ng / mL~0.91ng / mL, 0.910ng / mL~0.912ng / mL, 0.912ng / mL~0.915ng / mL, 0.915ng / mL~0.920ng / mL.

[0016] In some implementations, the fungus belongs to the subphylum Basidiomycota, class Agaricomycetes, order Phyllostachyales, and family Phyllostachyaceae.

[0017] In some embodiments, the fungus belongs to the subphylum Basidiomycota, class Agaricomycetes, order Phyllostachyales, family Phyllostachyaceae, and genus Phyllostachys.

[0018] In some embodiments, the fungus is Inonotus obliquus.

[0019] In some implementations, the fungus belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, and family Polyporaceae.

[0020] In some implementations, the fungus belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, family Polyporaceae, and genus Agaricus.

[0021] In some embodiments, the fungus is *Fomes fomentarius*.

[0022] In some implementations, the fungus belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Agaricales, and family Agaricalesaceae.

[0023] In some implementations, the fungus belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Agaricales, family Agaricaceae, and genus Agaricales.

[0024] In some embodiments, the fungus is Agaricus blazei Murill.

[0025] Inonotus obliquus, commonly known as birch polypore, belongs to the subphylum Basidiomycota, class Agaricomycetes, order Inonotales, family Inonotaceae, and genus Inonotus. It is a rare medicinal fungus, mainly distributed in high-altitude, cold regions between 40° and 50° north latitude, including Russia, Finland, Poland, Japan, and Heilongjiang and Jilin provinces in China.

[0026] Fomes fomentarius belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, family Polyporaceae, and genus Fomes. It mainly parasitizes the trunks of birch and oak trees, but also grows on the trunks of broad-leaved trees such as poplar, willow, alder, linden, and elm. Its fruit is known as wood hoof, birch fungus, fire velvet fomentarius, fire velvet fungus, wood purple fungus, tree base, etc., and is a traditional Chinese medicine.

[0027] The Brazilian mushroom (Agaricus blazei Murill) belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricaceae, order Agaricales, family Agaricaceae, genus Agaricus. Also known as Agaricus blazei, it is a valuable fungus used for both food and medicine.

[0028] In this invention, each cyclic dipeptide can be used in the form of a salt of an inorganic acid or organic acid, or a salt of an inorganic base or organic base. Such an acid or base may be selected depending on the application of the salt. Examples of inorganic acid salts include hydrochlorides, nitrates, sulfates, and methanesulfonates. Examples of organic acid salts include salts with dicarboxylic acids (such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid); and salts with monocarboxylic acids (such as acetic acid, propionic acid, and butyric acid). Examples of inorganic bases include hydroxides, carbonates, and bicarbonates of sodium, lithium, calcium, magnesium, aluminum, and ammonia. Examples of salts with organic bases include monoalkylamine salts, dialkylamine salts, or trialkylamine salts, such as salts of methylamine, dimethylamine, and triethylamine; monohydroxyalkylamine salts, dihydroxyalkylamine salts, or trihydroxyalkylamine salts; guanidine salts; and n-methylglucosamine salts.

[0029] In a second aspect, this application provides a method for preparing fungal fermentation products, the method comprising the following steps:

[0030] S1: Plate culture: Inoculate the fungal strain onto PDA medium and culture in a constant temperature and humidity incubator until the fungal growth is complete;

[0031] S2: Primary seed culture: The mycelium is inoculated into a seed culture medium and continuously cultured in a shaker to obtain primary seeds;

[0032] S3: Fermentation culture: The primary seed is inoculated into a shake flask containing fermentation medium and continuously fermented in a shaker. The resulting fermentation broth is centrifuged to remove the cells, and the supernatant is filtered through a filter membrane to obtain the fermentation filtrate.

[0033] In some implementations, the fungus belongs to the subphylum Basidiomycota, class Agaricomycetes, order Phyllostachyales, and family Phyllostachyaceae.

[0034] In some embodiments, the fungus belongs to the subphylum Basidiomycota, class Agaricomycetes, order Phyllostachyales, family Phyllostachyaceae, and genus Phyllostachys.

[0035] In some embodiments, the fungus is Inonotus obliquus.

[0036] In some implementations, the fungus belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, and family Polyporaceae.

[0037] In some implementations, the fungus belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, family Polyporaceae, and genus Agaricus.

[0038] In some embodiments, the fungus is *Fomes fomentarius*.

[0039] In some implementations, the fungus belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Agaricales, and family Agaricalesaceae.

[0040] In some implementations, the fungus belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Agaricales, family Agaricaceae, and genus Agaricales.

[0041] In some embodiments, the fungus is Agaricus blazei Murill.

[0042] In some implementations, in S1, the culture temperature is 27–29°C, for example, 28°C;

[0043] In some implementations, the culture time in S1 is 8 to 12 days, for example, 10 days.

[0044] In some implementations, S2 includes:

[0045] S2-1: Cut the mycelium into 1cm×1cm blocks and inoculate the blocks into seed culture medium; preferably, the ratio of the number of mycelium blocks to the volume of seed culture medium is 1:5mL (for example, take 10 blocks and inoculate them into 50mL of seed culture medium).

[0046] S2-2: Primary seeds are obtained by continuous culture on a shaker for 6 to 8 days (e.g., 7 days); preferably, the shaker culture conditions are 27 to 29°C (e.g., 28°C) and 160 to 200 rpm (e.g., 180 rpm).

[0047] In some embodiments, the seed culture medium is formulated as follows: 1000 mL deionized water, 2%–4% (e.g., 3%) glucose, 0.5%–1.5% (e.g., 1%) Nucel 845 mg (purchased from LESAFFRE), 0.05%–0.15% (e.g., 0.1%) potassium dihydrogen phosphate, and 0.04%–0.06% (e.g., 0.05%) anhydrous magnesium sulfate, at natural pH.

[0048] In some implementations, S3 includes:

[0049] S3-1: Inoculate the primary seeds into a shake flask containing fermentation medium at an inoculation rate of 4% to 6% (e.g., 5%). Preferably, the volume of the fermentation medium is 500 mL; more preferably, the volume of the shake flask is 2 L.

[0050] S3-2: Ferment continuously in a shaker for 6 to 8 days (e.g., 7); preferably, the fermentation conditions are 29 to 31°C (e.g., 30°C) and 140 to 160 rpm (e.g., 150 rpm).

[0051] In some implementations, the filter membrane used in S3 is a 0.22 μm filter membrane.

[0052] In some embodiments, the method further includes post-treatment of the fermentation filtrate.

[0053] In some implementations, the post-processing includes sterilization.

[0054] In some embodiments, the fermentation medium is formulated as follows: glucose 9 g / L to 11 g / L (e.g., 10 g / L), maltose 4 g / L to 6 g / L (e.g., 5 g / L), potato starch 30 g / L to 50 g / L (e.g., 40 g / L), Nucleol 845 MG 10 g / L to 20 g / L (e.g., 15 g / L), peptone 9 g / L to 11 g / L (e.g., 10 g / L), potassium dihydrogen phosphate 0.5 g / L to 1.5 g / L (e.g., 1 g / L), magnesium sulfate 0.4 g / L to 0.6 g / L (e.g., 0.5 g / L), (e.g., pH 5.5).

[0055] In some embodiments, the fungal fermentation product prepared by the method is the fungal fermentation product described in the first aspect of this application.

[0056] In a third aspect, this application provides a product comprising the fungal fermentation product as described in the first aspect above or the fungal fermentation product obtained by the preparation method described in the second aspect above.

[0057] In some embodiments, the product is a topical composition, such as a pharmaceutical composition or a cosmetic composition.

[0058] In addition to the fungal fermentation products described above, the cosmetic compositions of the present invention may optionally contain ingredients commonly used in skin care and cosmetic compositions, such as carriers, active ingredients, and excipients. Those skilled in the art can select the type and amount of these ingredients as needed.

[0059] The medium includes, for example, a diluent, a dispersant, or a carrier, examples of which include, but are not limited to, ethanol, dipropylene glycol, butanediol, etc.

[0060] The active ingredients include, for example, emollients, moisturizers, and anti-aging active ingredients.

[0061] Examples of the emollients include, but are not limited to, one or more of the following: olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, meadowfoam seed oil, safflower seed oil, rosehip oil, argan kernel oil, jojoba seed oil, sunflower seed oil, murrilla palm oil, squalane, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutylene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, lauroyl sarcosinate isopropyl, isononyl isononanoate, hydrogenated polydecene, triglycerides (ethylhexanoate), cetyl ethylhexanoate, bis-diethoxydiethylene cyclohexane 1,4-dicarboxylic acid ester, caprylic / capric triglyceride, oleyl erucic acid ester, octyl dodecyl myristate, octyl dodecyl alcohol, polydimethylsiloxane, octyl polymethylsiloxane, cetyl polydimethylsiloxane, cyclopentamethoxydimethylsiloxane, etc. Examples of solid emollients include, but are not limited to, one or more of the following: cetyl alcohol, stearyl alcohol, cetearyl alcohol, behenyl alcohol, squalene, lauric acid, myristic acid, palmitic acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ceresin, jojoba seed wax, paraffin wax, microcrystalline wax, hydrogenated rice bran wax, hydrogenated coconut oil glycerides, glyceryl behenate / eicosyl ester, myristic acid myristicate, bis-diglyceride polyacryladiate-2, shea butter, and muruman seed butter.

[0062] Examples of the moisturizers include, but are not limited to, one or more of the following: glycerin, diglycerin, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, 1,2-pentanediol, polyethylene glycol-8, polyethylene glycol-32, methyl glucetol polyether-10, methyl glucetol polyether-20, PEG / PPG-17 / 6 copolymer, glycerin polyether-7, glycerin polyether-26, glyceryl glucoside, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerin, sucrose, trehalose, rhamnose, mannose, raffinose, betaine, erythritol, xylitol, urea, glyceryl polyether-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, acetylated sodium hyaluronate, sodium polyglutamate, hydrolyzed sclerotium gum, budding short stem enzyme polysaccharide, tremella polysaccharide, and soybean seed polysaccharide.

[0063] Examples of the anti-aging active ingredients include, but are not limited to, tocopherol (vitamin E), retinol, retinyl palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin, ellagic acid, ubiquinone, whey protein, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicylyl phytosphingosine, concentrated birch sap, silymarin, silk fibroin, sodium tocopheryl phosphate, ribonucleic acid (RNA), dipeptide diaminobutyryl benzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide-7, grape (VITIS VINIFERA) seed extract, rosewood (PTEROCARPUS MARSUPIUM) bark extract, tea (CAMELLIA SINENSIS) polyphenols, wine extract, apple seed extract, European beech (FAGUS SYLVATICA) bud extract, and hydrolyzed baobab (ADANSONIA) The ingredients include one or more of the following: Digitata extract, Artemisia extract, Iris florentina root extract, hesperidin, ginsenosides, Salvia miltiorrhizina extract, nicotinamide, ursolic acid, sodium hyaluronate, acetylated sodium hyaluronate, hydrolyzed sodium hyaluronate, lycopene, coffee (Coffea arabica) extract, dipeptide-2, lactic acid, superoxide dismutase (SOD), evening primrose (Oenothera biennes) oil, ceramide, dipalmitoyl hydroxyproline, hydroxystearic acid, salicylic acid, ergothioneine, lysophosphatidylcholine, carnosine, decarboxylated carnosine HCl, lipoic acid, adenosine, glycogen, resveratrol, ferulic acid, Bifida ferment lysate, and lactic acid bacteria ferment lysate.

[0064] The excipients include, for example, emulsifiers, thickeners, preservatives, and fragrances.

[0065] Examples of such emulsifiers include, but are not limited to, cetearyl oleate, sorbitan 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 polyglycerol-2 stearate, PEG-60 hydrogenated castor oil, stearyl oleate-2, stearyl oleate-21, PPG-13-decyltetradecyl alcohol polyether-24, cetearyl glucoside, PEG-100 stearate, and glycerin. Stearate, glyceryl stearate SE, cocoyl glucoside, cetearyl alcohol polyether-25, PEG-40 stearate, polyglycerol-3-methylglucose distearate, glyceryl stearate citrate, polyglycerol-10 stearate, polyglycerol-10 myristate, polyglycerol-10 dioleate, polyglycerol-10 laurate, polyglycerol-10 isostearate, polyglycerol-10 oleate, polyglycerol-10 diisostearate, polyglycerol-6 laurate, polyglycerol-6 myristate, sucrose stearate, sucrose polystearate, etc.

[0066] Examples of such thickeners include, but are not limited to, one or more of the following polymers: carbomers, acrylates and their derivatives, xanthan gum, gum arabic, polyethylene glycol-14M, polyethylene glycol-90M, succinyl polysaccharide, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

[0067] Examples of the preservatives include, but are not limited to, one or more of the following: methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis(hydroxymethyl)imidazolidinyl urea, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, capryloyl hydroxamic acid, 1,2-hexanediol, 1,2-pentanediol, p-hydroxyacetophenone, caprylyl glycol, glyceryl caprylate, undecenoic acid glyceride, sorbitan caprylate, ethylhexylglycerin, and peony root extract.

[0068] The cosmetic composition of the present invention can be prepared by any suitable method known in the art. As needed, the cosmetic composition can be formulated into various dosage forms such as ointments, creams, lotions, and serums.

[0069] In some embodiments, the topical composition of the present invention is a pharmaceutical composition that can be used to treat skin diseases, particularly skin diseases related to cellular aging. The topical pharmaceutical composition can be formulated into dosage forms such as ointments, gels, foams, creams, and sprays.

[0070] In other embodiments, the product is a medical aesthetic product. In this invention, medical aesthetics refers to the repair and reshaping of a person's appearance and the shape of various parts of the body using drugs, surgery, medical devices, and other invasive or irreversible medical techniques. Medical aesthetic products can be formulated into suitable dosage forms, such as injections, transdermal drug delivery preparations (e.g., microneedles, patches), etc.

[0071] In a fourth aspect, this application provides the use of fungal fermentation products as described in the first aspect above or fungal fermentation products obtained by the preparation method described in the second aspect above for the preparation of products that slow down skin aging.

[0072] In some embodiments, the reduction of skin aging includes reducing wrinkles and / or firming the skin. Reducing wrinkles may include reducing the area, volume, and / or number of wrinkles, or lowering the severity of wrinkles. The area, volume, and / or number of wrinkles can be measured using an instrument (e.g., a skin wrinkle analyzer). The severity of wrinkles can be assessed by a professional according to certain standards (e.g., a wrinkle assessment scale). In some embodiments, the wrinkles include one or more of forehead wrinkles, crow's feet, under-eye wrinkles, and nasolabial folds.

[0073] In a fifth aspect, this application provides the use of a cyclic dipeptide or its salt in the preparation of a product for slowing down skin aging, wherein the cyclic dipeptide is cyclic proline-leucine, or the cyclic dipeptide comprises 2, 3, 4, 5 or 6 of the following: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0074] In some embodiments, the slowing down of skin aging includes reducing wrinkles and / or firming the skin. In some embodiments, reducing wrinkles includes reducing the area, volume, and / or number of wrinkles, or lowering the severity of wrinkles. In some embodiments, the wrinkles include one or more of forehead wrinkles, crow's feet, under-eye wrinkles, and nasolabial folds.

[0075] In this invention, the amino acid forming the cyclic dipeptide can be an L-amino acid, a D-amino acid, or a racemic mixture of these two configurations, such as L-proline, L-tyrosine, L-isoleucine, L-leucine, L-phenylalanine, L-valine, D-proline, D-tyrosine, D-isoleucine, D-leucine, D-phenylalanine, D-valine, DL-proline, DL-tyrosine, DL-isoleucine, DL-leucine, DL-phenylalanine, and DL-valine.

[0076] The inventors have discovered that some cyclic dipeptides, as well as the fungal fermentation products of the present invention containing cyclic dipeptides, can reduce the activity of SA-β-gal in cells, and therefore can be used for disease prevention or treatment or for skin care or medical aesthetics.

[0077] Therefore, in a sixth aspect, this application provides the use of fungal fermentation products as described in the first aspect above or fungal fermentation products obtained by the preparation method described in the second aspect above for the preparation of products used to reduce the activity of SA-β-gal in cells.

[0078] In a seventh aspect, this application provides the use of a cyclic dipeptide or a salt thereof for preparing a product for reducing the activity of SA-β-gal in cells; wherein the cyclic dipeptide is cyclic isoleucine-proline, cyclic proline-leucine, or cyclic leucine-leucine, or the cyclic dipeptide comprises 2, 3, 4, 5, or 6 of the following: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0079] SA-β-Gal (Senescence-Associated β-Galactosidase) is a β-galactosidase that exhibits increased activity during cellular senescence. SA-β-Gal is known to accumulate in aging skin and muscle tissue and is associated with diseases such as chronic liver disease.

[0080] In some embodiments, the products described in aspects four through seven are topical compositions, such as pharmaceutical or cosmetic compositions. The cosmetic compositions may also optionally contain ingredients commonly used in skincare cosmetic compositions described above, such as carriers, active ingredients, and excipients, the types and amounts of which can be selected by those skilled in the art as needed. Pharmaceutical compositions can be used to treat skin diseases, particularly those related to SA-β-Gal. Topical pharmaceutical compositions can be formulated into dosage forms such as ointments, gels, foams, creams, and sprays.

[0081] In other embodiments, such as those described in aspects four through seven, the products are medical aesthetic products and can be formulated into suitable dosage forms, such as injections, transdermal drug delivery preparations (e.g., microneedles, patches), etc.

[0082] In some embodiments, the cyclic dipeptides described in aspects four through seven include cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0083] In some embodiments, the cyclic dipeptides described in aspects four through seven comprise equal masses of cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0084] In some embodiments, the product of the present invention can be used externally, for example, by applying it to the skin surface through a topical application. In other embodiments, the product of the present invention can be used internally, for example, by injection, transdermal administration, or other methods.

[0085] In some embodiments, the product of the present invention is used for non-therapeutic purposes.

[0086] The present invention also provides a method for reducing SA-β-Gal activity in cells, comprising administering an effective amount of a cyclic dipeptide to the cells; wherein the cyclic dipeptide is cyclic isoleucine-proline, cyclic proline-leucine, or cyclic leucine-leucine, or the cyclic dipeptide comprises 2, 3, 4, 5, or 6 of the following: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0087] In some embodiments, the method is used in vivo, for example, when the cells are cells in the body of a subject (e.g., mammals; such as bovine, equine, suidae, canine, feline, rodent, primate; e.g., human); or, the method is used in vitro, for example, when the cells are cells in vitro (e.g., cell lines or cells derived from the subject).

[0088] In some implementations, the cells are fibroblasts.

[0089] In some implementations, the method is used for non-therapeutic purposes (e.g., for scientific research).

[0090] In some embodiments, the cyclic dipeptides applied by the method include cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

[0091] In some embodiments, the cyclic dipeptide applied by the method comprises equal masses of cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine. Attached Figure Description

[0092] Figure 1 The effect of fermentation filtrate on zebrafish embryo viability was shown.

[0093] Figure 2 The effects of fermentation filtrate and cyclic dipeptide on the recovery rate of zebrafish tail fins were shown.

[0094] Explanation of the source of biological materials

[0095] The *Inonotus obliquus* species involved in this invention were collected from Mohe, Heilongjiang Province, and identified as *Inonotus obliquus* through laboratory isolation, purification, and identification. The *Fomes fomentarius* species were collected from Yunnan Province and identified as *Agaricus blazei* Murill through laboratory isolation, purification, and identification.

[0096] Instructions on the Preservation of Biological Materials

[0097] This invention relates to the following biological materials that have been deposited at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing):

[0098] The Inonotus obliquus strain YST02 has the accession number CGMCC No.20206 and the accession date is June 17, 2020. Detailed Implementation

[0099] The present invention will be further described in detail below with reference to the embodiments. However, it should be understood that these embodiments and comparative examples are merely for illustrating the present invention in more detail, and should not be construed as limiting the scope of the appended claims in any way.

[0100] Example 1: Preparation of Inonotus obliquus fermentation filtrate

[0101] The preparation of fermentation filtrate is divided into several stages, including plate culture, primary seed culture, and fermentation culture.

[0102] (1) Plate culture: The frozen Inonotus obliquus strain was inoculated onto PDA medium and cultured in a constant temperature and humidity incubator at 28℃ for 10 days until the mycelium was fully grown.

[0103] (2) Primary seed culture: Cut the mycelium on the solid plate into 1cm×1cm small mycelium blocks, take 10 blocks and inoculate them into 50ml seed culture medium, and culture them continuously on a shaker for 7 days to obtain primary seeds. The shaker culture conditions are 28℃ and 180rpm.

[0104] (3) Fermentation culture: The primary seed was inoculated into a 2L shake flask containing 500mL of fermentation medium at a 5% inoculation rate, and fermented continuously in a shaker for 7 days at 30℃ and 150rpm. The obtained fermentation broth was centrifuged to remove the main cells, and the supernatant was filtered through a 0.22μm filter membrane to obtain the fermentation filtrate, and the content of cyclic dipeptides was determined.

[0105] The seed culture medium formula was as follows: 1000 mL deionized water, 3% glucose, 1% Nucel 845 MG (purchased from LESAFFRE), 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, at natural pH.

[0106] The fermentation medium formula is as follows: glucose 10g / L, maltose 5g / L, potato starch 40g / L, Nucleol 845MG 15g / L, peptone 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, pH 5.5.

[0107] The method for determining the content of cyclic dipeptides was as follows: based on the content of cyclic dipeptide standards, liquid chromatography-mass spectrometry (LC-MS / MS) was used for determination. The results are shown in Table 1.

[0108] Example 2: Preparation of Fermentation Filtrate of *Pogostemon cablin*

[0109] The preparation of fermentation filtrate is divided into several stages, including plate culture, primary seed culture, and fermentation culture.

[0110] (1) Plate culture: The frozen Porphyra yezoensis strain was inoculated onto PDA medium and cultured in a constant temperature and humidity incubator at 28℃ for 10 days until the mycelium was fully grown.

[0111] (2) Primary seed culture: Cut the mycelium on the solid plate into 1cm×1cm small mycelium blocks, take 10 blocks and inoculate them into 50ml seed culture medium, and culture them continuously on a shaker for 7 days to obtain primary seeds. The shaker culture conditions are 28℃ and 180rpm.

[0112] (3) Fermentation culture: The primary seed was inoculated into a 2L shake flask containing 500mL of fermentation medium at a 5% inoculation rate, and fermented continuously in a shaker for 7 days at 30℃ and 150rpm. The obtained fermentation broth was centrifuged to remove the main cells, and the supernatant was filtered through a 0.22μm filter membrane to obtain the fermentation filtrate, and the content of cyclic dipeptides was determined.

[0113] The seed culture medium formula was as follows: 1000 mL deionized water, 3% glucose, 1% Nucel 845 MG (purchased from LESAFFRE), 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, at natural pH.

[0114] The fermentation medium formula is as follows: glucose 10g / L, maltose 5g / L, potato starch 40g / L, Nucleol 845MG 15g / L, peptone 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, pH 5.5.

[0115] The method for determining the content of cyclic dipeptides was as follows: based on the content of cyclic dipeptide standards, liquid chromatography-mass spectrometry (LC-MS / MS) was used for determination. The results are shown in Table 1.

[0116] Example 3: Preparation of Fermentation Filtrate from Agaricus blazei

[0117] The preparation of fermentation filtrate is divided into several stages, including plate culture, primary seed culture, and fermentation culture.

[0118] (1) Plate culture: The frozen Brazilian mushroom spawn was inoculated onto PDA medium and cultured in a constant temperature and humidity incubator at 28℃ for 10 days until the mycelium was fully grown.

[0119] (2) Primary seed culture: Cut the mycelium on the solid plate into 1cm×1cm small mycelium blocks, take 10 blocks and inoculate them into 50ml seed culture medium, and culture them continuously on a shaker for 7 days to obtain primary seeds. The shaker culture conditions are 28℃ and 180rpm.

[0120] (3) Fermentation culture: The primary seed was inoculated into a 2L shake flask containing 500mL of fermentation medium at a 5% inoculation rate, and fermented continuously in a shaker for 7 days at 30℃ and 150rpm. The obtained fermentation broth was centrifuged to remove the main cells, and the supernatant was filtered through a 0.22μm filter membrane to obtain the fermentation filtrate, and the content of cyclic dipeptides was determined.

[0121] The seed culture medium formula was as follows: 1000 mL deionized water, 3% glucose, 1% Nucel 845 MG (purchased from LESAFFRE), 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, at natural pH.

[0122] The fermentation medium formula is as follows: glucose 10g / L, maltose 5g / L, potato starch 40g / L, Nucleol 845MG 15g / L, peptone 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, pH 5.5.

[0123] The method for determining the content of cyclic dipeptides was as follows: based on the content of cyclic dipeptide standards, liquid chromatography-mass spectrometry (LC-MS / MS) was used for determination. The results are shown in Table 1.

[0124] Table 1. Cyclic dipeptide content in fermentation filtrate

[0125]

[0126] Example 4: Detection of embryotoxicity of Inonotus obliquus fermentation filtrate

[0127] Select 2-day-fiber zebrafish embryos with normal development and place them into a six-well cell culture plate, 15 fish per well. Remove the standard dilution water from the six-well plate without harming the juvenile fish, and quickly add 3 mL of the corresponding concentration of the test substance dilution to each well.

[0128] After thorough mixing, wrap the six-well plate with aluminum foil and incubate in a biochemical incubator in the dark for 2 hours. Then, place it in a UV irradiator for further incubation, followed by another 18 hours (3 days post-flop) in the biochemical incubator before observation. Record the proportion of zebrafish that did not die (no heartbeat) and exhibited other toxic effects (pericardial edema, trunk bending, unresponsiveness to mechanical stimulation, unclear muscle texture, etc.). Results are shown below. Figure 1 The results in the figure show that the fermentation filtrate of Inonotus obliquus is non-toxic to embryos.

[0129] Example 5: Detection of embryotoxicity of Fermentation Filtrate of *Pogostemon cablin*

[0130] Select 2-day-fiber zebrafish embryos with normal development and place them in a six-well cell culture plate, 15 embryos per well. Remove the standard dilution water from the six-well plate without harming the embryos, and quickly add 3 mL of the corresponding concentration of the test substance dilution to each well.

[0131] After thorough mixing, wrap the six-well plate with aluminum foil and incubate in a biochemical incubator in the dark for 2 hours. Then, place it in a UV irradiator for further incubation in the biochemical incubator for 18 hours (3 days post-flop) before observation. Record the proportion of zebrafish that did not die (no heartbeat) or exhibit other toxic effects (pericardial edema, trunk bending, unresponsiveness to mechanical stimulation, unclear muscle texture, etc.). Results are shown below. Figure 1 The results in the figure show that the fermentation filtrate of *Pheretima aspergillum* is non-toxic to the embryo.

[0132] Example 6: Detection of embryotoxicity of Agaricus blazei fermentation filtrate

[0133] Select 2-day-fiber zebrafish embryos with normal development and place them in a six-well cell culture plate, 15 embryos per well. Remove the standard dilution water from the six-well plate without harming the embryos, and quickly add 3 mL of the corresponding concentration of the test substance dilution to each well.

[0134] After thorough mixing, wrap the six-well plate with aluminum foil and incubate in a biochemical incubator in the dark for 2 hours. Then, place it in a UV irradiator for further incubation in the biochemical incubator for 18 hours (3 days post-flop) before observation. Record the proportion of zebrafish that did not die (no heartbeat) or exhibit other toxic effects (pericardial edema, trunk bending, unresponsiveness to mechanical stimulation, unclear muscle texture, etc.). Results are shown below. Figure 1The results in the figure show that the fermentation filtrate of *Agaricus blazei* is non-toxic to embryos.

[0135] Example 7: Detection of Embryotoxicity of Cyclic Dipeptides

[0136] Normally developed zebrafish embryos at 2 dpf were selected and placed in six-well cell culture plates, 15 embryos per well. Without harming the embryos, the standard dilution water in the six-well plate was removed, and 3 mL of the corresponding concentration of the test substance dilution was quickly added to each well. The test substances were the cyclic dipeptide standards shown in Table 2.

[0137] After thorough mixing, the six-well plate was wrapped with aluminum foil and incubated in a biochemical incubator in the dark for 2 hours. Then, it was placed in a UV irradiator for further incubation in the biochemical incubator for 18 hours (3 days post-conversion) before observation. The proportion of zebrafish that did not die (no heartbeat) or exhibit other toxic effects (pericardial edema, trunk bending, unresponsiveness to mechanical stimulation, unclear muscle texture, etc.) was recorded. The results are shown in Table 2. The results in the table show that the cyclic dipeptide is not toxic to embryos.

[0138] Table 2. Effects of cyclic dipeptide on zebrafish embryo viability.

[0139]

[0140]

[0141] Example 8: Anti-wrinkle effect of Inonotus obliquus fermentation filtrate on zebrafish

[0142] Group according to the following method:

[0143] Blank control group: containing zebrafish juveniles and working solution, without modeling.

[0144] Model control group: containing zebrafish juveniles and working solution, and subjected to ultraviolet irradiation to establish the model.

[0145] Positive control group: containing positive sample tea polyphenols and zebrafish juveniles, which were subjected to ultraviolet irradiation to create a model.

[0146] Test group: containing the test substance and zebrafish juveniles, the model was created by ultraviolet irradiation, and different concentration groups of the test substance were set.

[0147] Experimental process

[0148] Normally developed 2-day-fif (dpf) zebrafish juveniles were selected and randomly assigned to six-well plates, 15 fish per well. Without harming the juveniles, the standard dilution water in the six-well plates was removed, and 3 mL of the corresponding concentration of the test substance dilution was quickly added to each well. After thorough mixing, the six-well plates were wrapped with aluminum foil and incubated in a (28.5℃±1.0℃) biochemical incubator in the dark for 2 hours. Then, they were placed in a UV irradiator and irradiated with the following conditions for 10 min: UVA: 1.30 mW / cm², UVB: 0.38 mW / cm². Afterward, they were incubated in a (28.5℃±1.0℃) biochemical incubator in the dark for another 18 hours (3 dpf). After incubation, zebrafish were transferred to 1.5 mL EP tubes, and the E3 buffer was aspirated. 1 mL of 4% paraformaldehyde was added, and the tubes were fixed at room temperature for at least 30 minutes. Then, the tubes were fixed with 3% methylcellulose. The zebrafish were observed and photographed under a stereomicroscope. During photography, the zebrafish should have their heads facing left, sides down, and bodies horizontal. All zebrafish photographs must be taken under the same instrument and environmental conditions, and the zebrafish positions should be consistent. After photography, the images were analyzed using ImageJ image analysis software, with the caudal fin as the quantitative region. The analysis parameter was set to area, and data were obtained from the images. Ten valid data points were taken from each group. The recovery rate was calculated based on the caudal fin area; a higher recovery rate indicates stronger wrinkle resistance of the test substance.

[0149] The test results are as follows Figure 2 This indicates that the fermentation filtrate of Inonotus obliquus has a significant anti-wrinkle effect on zebrafish.

[0150] Example 9: Anti-wrinkle effect of Fermentation Filtrate of *Pogostemon cablin* on Zebrafish

[0151] Group according to the following method:

[0152] Blank control group: containing zebrafish juveniles and working solution, without modeling.

[0153] Model control group: containing zebrafish juveniles and working solution, and subjected to ultraviolet irradiation to establish the model.

[0154] Positive control group: containing positive sample tea polyphenols and zebrafish juveniles, which were subjected to ultraviolet irradiation to create a model.

[0155] Test group: containing the test substance and zebrafish juveniles, the model was created by ultraviolet irradiation, and different concentration groups of the test substance were set.

[0156] Experimental process

[0157] Normally developed 2-day-fif (dpf) zebrafish juveniles were selected and randomly assigned to six-well plates, 15 fish per well. Without harming the juveniles, the standard dilution water in the six-well plates was removed, and 3 mL of the corresponding concentration of the test substance dilution was quickly added to each well. After thorough mixing, the six-well plates were wrapped with aluminum foil and incubated in a (28.5℃±1.0℃) biochemical incubator in the dark for 2 hours. Then, they were placed in a UV irradiator and irradiated with the following conditions for 10 min: UVA: 1.30 mW / cm², UVB: 0.38 mW / cm². Afterward, they were incubated in a (28.5℃±1.0℃) biochemical incubator in the dark for another 18 hours (3 dpf). After incubation, zebrafish were transferred to 1.5 mL EP tubes, and the E3 buffer was aspirated. 1 mL of 4% paraformaldehyde was added, and the tubes were fixed at room temperature for at least 30 minutes. Then, the tubes were fixed with 3% methylcellulose. The zebrafish were observed and photographed under a stereomicroscope. During photography, the zebrafish should have their heads facing left, sides down, and bodies horizontal. All zebrafish photographs must be taken under the same instrument and environmental conditions, and the zebrafish positions should be consistent. After photography, the images were analyzed using ImageJ image analysis software, with the caudal fin as the quantitative region. The analysis parameter was set to area, and data were obtained from the images. Ten valid data points were taken from each group. The recovery rate was calculated based on the caudal fin area; a higher recovery rate indicates stronger wrinkle resistance of the test substance.

[0158] The test results are as follows Figure 2 This indicates that the fermentation filtrate of *Porphyra yezoensis* has a significant anti-wrinkle effect on zebrafish.

[0159] Example 10: Anti-wrinkle effect of Brazilian mushroom fermentation filtrate on zebrafish

[0160] Group according to the following method:

[0161] Blank control group: containing zebrafish juveniles and working solution, without modeling.

[0162] Model control group: containing zebrafish juveniles and working solution, and subjected to ultraviolet irradiation to establish the model.

[0163] Positive control group: containing positive sample tea polyphenols and zebrafish juveniles, which were subjected to ultraviolet irradiation to create a model.

[0164] Test group: containing the test substance and zebrafish juveniles, the model was created by ultraviolet irradiation, and different concentration groups of the test substance were set.

[0165] Experimental process

[0166] Normally developed 2-day-fifth (dpf) zebrafish juveniles were selected and randomly assigned to six-well plates, 15 fish per well. Without harming the juveniles, the standard dilution water in the six-well plates was removed, and 3 mL of the corresponding concentration of the test substance dilution was quickly added to each well. After thorough mixing, the six-well plates were wrapped with aluminum foil and incubated in a biochemical incubator at (28.5℃±1.0℃) in the dark for 2 hours. Afterward, they were placed in a UV irradiator and irradiated with UVA at the following conditions: 10 min UVA: 1.30 mW / cm². 2 UVB: 0.38mW / cm 2 Afterwards, the zebrafish were incubated in a biochemical incubator at (28.5℃±1.0℃) in the dark for 18 hours (3 dpf). After incubation, the zebrafish were transferred to 1.5 mL EP tubes, the E3 buffer was aspirated, and 1 mL of 4% paraformaldehyde was added. The tubes were fixed at room temperature for at least 30 minutes, then fixed with 3% methylcellulose. The zebrafish were observed and photographed under a stereomicroscope. During photography, the zebrafish should have their heads facing left, sides down, and bodies horizontal. All zebrafish photographs must be taken under the same instrument and environmental conditions, and the zebrafish positions should be consistent. After photography, the images of the zebrafish were analyzed using ImageJ software, with the caudal fin as the quantitative region. The analysis parameter was set to area, and data were obtained from the images. Ten valid data points were taken from each group. The recovery rate was calculated based on the caudal fin area; a higher recovery rate indicates stronger wrinkle resistance of the test substance.

[0167] The test results are as follows Figure 2 This indicates that the fermented filtrate of Brazilian mushrooms has a significant anti-wrinkle effect on zebrafish.

[0168] Example 11: Anti-wrinkle effect of cyclic dipeptide on zebrafish

[0169] Group according to the following method:

[0170] Blank control group: containing zebrafish juveniles and working solution, without modeling.

[0171] Model control group: containing zebrafish juveniles and working solution, and subjected to ultraviolet irradiation to establish the model.

[0172] Positive control group: containing positive sample tea polyphenols and zebrafish juveniles, which were subjected to ultraviolet irradiation to create a model.

[0173] Test group: Zebrafish juveniles were irradiated with a mixture of cyclic dipeptides to induce a model. Different concentration groups of the test substance were set up. The cyclic dipeptide mixture used consisted of cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine, all at a concentration of 0.1 ppm.

[0174] Experimental process

[0175] Normally developed 2-day-fif (dpf) zebrafish juveniles were selected and randomly assigned to six-well plates, 15 fish per well. Without harming the juveniles, the standard dilution water in the six-well plates was removed, and 3 mL of the corresponding concentration of the test substance dilution was quickly added to each well. After thorough mixing, the six-well plates were wrapped with aluminum foil and incubated in a (28.5℃±1.0℃) biochemical incubator in the dark for 2 hours. Then, they were placed in a UV irradiator and irradiated with the following conditions for 10 min: UVA: 1.30 mW / cm², UVB: 0.38 mW / cm². Afterward, they were incubated in a (28.5℃±1.0℃) biochemical incubator in the dark for another 18 hours (3 dpf). After incubation, zebrafish were transferred to 1.5 mL EP tubes, and the E3 buffer was aspirated. 1 mL of 4% paraformaldehyde was added, and the tubes were fixed at room temperature for at least 30 minutes. Then, the tubes were fixed with 3% methylcellulose. The zebrafish were observed and photographed under a stereomicroscope. During photography, the zebrafish should have their heads facing left, sides down, and bodies horizontal. All zebrafish photographs must be taken under the same instrument and environmental conditions, and the zebrafish positions should be consistent. After photography, the images were analyzed using ImageJ image analysis software, with the caudal fin as the quantitative region. The analysis parameter was set to area, and data were obtained from the images. Ten valid data points were taken from each group. The recovery rate was calculated based on the caudal fin area; a higher recovery rate indicates stronger wrinkle resistance of the test substance.

[0176] The test results are as follows Figure 2 This indicates that the cyclic dipeptide mixture has a significant anti-wrinkle effect on zebrafish.

[0177] Example 12: Effect of cyclic dipeptide on SA-β-gal in senescent fibroblasts

[0178] Fibroblasts from passage 15 were selected and divided into: ① Normal control group: 2 mL of serum-free DMEM medium; ② Model group: 2 mL of serum-free DMEM medium (containing 50 μM t-BHP); ③ Positive control group: 2 mL of serum-free DMEM medium (containing 200 U / mL catalase); ④ Cyclic proline-tyrosine group; ⑤ Cyclic isoleucine-proline group; ⑥ Cyclic proline-leucine group; ⑦ Cyclic proline-phenylalanine group; ⑧ Cyclic valine-phenylalanine group; ⑨ Cyclic leucine-leucine group. The concentration of each cyclic dipeptide was 0.1 ppm. After one hour of culture, each group was replaced with DMEM medium containing 5% (v / v) serum and cultured at 37°C for 24 hours. ⑩ The mixed cyclic dipeptide group used a mixture of cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine, all with a concentration of 0.1 ppm.

[0179] After repeating cell modeling and cell recovery twice, cells in each group were digested with trypsin, and digestion was terminated with 1 mL of DMEM medium containing 5% (v / v) serum. 500 μL of each cell was seeded into new 6-well plates, and 1.5 mL of DMEM medium containing 5% (v / v) serum was added. Staining was performed using the Beyotime Cell Senescence β-Galactosidase Staining Kit (C0602), and observation was performed under a light microscope.

[0180] Data analysis revealed that the positive rate of SA-β-gal in the normal control group was 10.6%, while the positive rate in the model group was 35.2%, and the positive rate of SA-β-gal in the positive drug group was 16.1%, indicating successful model establishment. The positive rates of SA-β-gal in the cyclic proline-tyrosine group were 34.1%, cyclic isoleucine-proline group was 26.1%, cyclic proline-leucine group was 24.7%, cyclic proline-phenylalanine group was 32.3%, cyclic valine-phenylalanine group was 32.7%, cyclic leucine-leucine group was 17.5%, and the positive rate in the mixed cyclic dipeptide group was 21.2%. This indicates that cyclic isoleucine-proline, cyclic proline-leucine, cyclic leucine-leucine, and mixed cyclic dipeptides have anti-wrinkle effects.

[0181] Example 13: Anti-wrinkle effects of cyclic dipeptides in humans

[0182] The products from Examples 1, 2, and 3, along with the cyclic dipeptide mixture, were used as the test group for volunteer testing.

[0183] The cyclic dipeptide mixture used consisted of cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine, all with a concentration of 0.1 ppm.

[0184] Healthy Chinese women aged 30-55 were selected as participants. Their mean F4 score on both cheeks was >6, or their mean R2 score was ≤0.65. Experts assessed their crow's feet (2-4), under-eye wrinkles (2-5), and nasolabial folds (1-3) according to the *Skin Aging Atlas Volume 2 Asian Type*. Informed consent was obtained from all participants. Volunteers were randomly divided into four groups of 35 each. One group used *Inonotus obliquus* fermentation filtrate, one group used *Inonotus ciliatus* fermentation filtrate, one group used *Agaricus brasiliensis* fermentation filtrate, and one group used a cyclic dipeptide mixture. Use was continued for 28 days, applied once daily, morning and evening. The volume of crow's feet, under-eye wrinkles, and nasolabial folds was measured before and after product use using a PRIMOS-CR instrument. Experts were invited to evaluate the volunteers' forehead wrinkle levels, crow's feet wrinkle levels, under-eye wrinkle levels, and nasolabial fold levels before and after product use. The evaluation of the product's anti-wrinkle and firming effects was collected from volunteers through questionnaires.

[0185] The test results are shown in Table 3.

[0186] Table 3 Evaluation results of anti-wrinkle efficacy in humans

[0187]

[0188]

[0189] Results Analysis: The 28-day continuous human trial results showed that the fermented filtrate of *Inonotus obliquus*, *Inonotus truncatus*, *Inonotus africanus*, and the cyclic dipeptide mixture had significant effects. Instrumental measurements indicated that the experimental group showed significant improvement in nasolabial folds, crow's feet, under-eye wrinkles, and forehead wrinkles, demonstrating that the fermented filtrate of *Inonotus obliquus*, *Inonotus truncatus*, *Inonotus africanus*, and the cyclic dipeptide mixture has good anti-wrinkle and firming effects.

[0190] The technical solutions described above are preferred embodiments of the present invention. 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 to be within the protection scope of the present invention.

Claims

1. A fungal fermentation product comprising one or more of the following cyclic dipeptides or their salts: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, or cyclic leucine-leucine. Preferably, the fungal product is a fungal fermentation filtrate, which contains one or more cyclic dipeptides or their salts in the following amounts: 0.15 ng / mL to 1.8 ng / mL cyclic proline-tyrosine, 2.0 ng / mL to 6.1 ng / mL cyclic isoleucine-proline, 2.5 ng / mL to 48.7 ng / mL cyclic proline-leucine, 2.0 ng / mL to 8.0 ng / mL cyclic proline-phenylalanine, 0.06 ng / mL to 0.39 ng / mL cyclic valine-phenylalanine, and 0.05 ng / mL to 0.92 ng / mL cyclic leucine-leucine.

2. A method for preparing fungal fermentation products, the method comprising the following steps: S1: Plate culture: Inoculate the fungal strain onto PDA medium and culture in a constant temperature and humidity incubator until the fungal growth is complete; S2: Primary seed culture: The mycelium is inoculated into a seed culture medium and continuously cultured in a shaker to obtain primary seeds; S3: Fermentation culture: The primary seed is inoculated into a shake flask containing fermentation medium and continuously fermented in a shaker. The resulting fermentation broth is centrifuged to remove the cells, and the supernatant is filtered through a filter membrane to obtain the fermentation filtrate.

3. The method of claim 2, wherein the fungus belongs to the subphylum Basidiomycota, class Agaricomycetes, order Phyllostachyales, family Phyllostachyaceae; preferably, the fungus belongs to the subphylum Basidiomycota, class Agaricomycetes, order Phyllostachyales, family Phyllostachyaceae, genus Phyllostachys; preferably, the fungus is Inonotus obliquus. Alternatively, the fungus belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, and family Polyporaceae; preferably, the fungus belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, and family Polyporaceae, genus Fomesfomentarius; preferably, the fungus is Fomesfomentarius. Alternatively, the fungus belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Agaricales, family Agaricales; preferably, the fungus belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Agaricales, family Agaricales, genus Agaricus; preferably, the fungus is Agaricus blazei Murill.

4. The method of claim 2 or 3, wherein, S1 has one or more of the following characteristics: (1) In S1, the culture temperature is 27-29℃; (2) In S1, the culture time is 8 to 12 days.

5. The method according to any one of claims 2 to 4, wherein, S2 includes: S2-1: Cut the mycelium into 1cm×1cm blocks and inoculate the blocks into seed culture medium; preferably, the ratio of the number of mycelium blocks to the volume of seed culture medium is 1:5mL. S2-2: First-grade seeds are obtained by continuous culture on a shaker for 6 to 8 days; preferably, the shaker culture conditions are 27 to 29°C and 160 to 200 rpm.

6. The method according to any one of claims 2 to 5, wherein, S3 includes: S3-1: Inoculate the primary seeds into a shake flask containing fermentation medium at an inoculation rate of 4% to 6%. Preferably, the volume of the fermentation medium is 500 mL; more preferably, the volume of the shake flask is 2 L. S3-2: Ferment continuously in a shaker for 6 to 8 days; preferably, the fermentation conditions are 29 to 31°C and 140 to 160 rpm.

7. The method according to any one of claims 2 to 6, wherein, The filter membrane used in S3 is a 0.22μm filter membrane; Preferably, the method further includes: post-treatment of the fermentation filtrate; Preferably, the post-processing includes sterilization.

8. A product comprising the fungal fermentation product of claim 1 or the fungal fermentation product prepared by the method of any one of claims 2 to 7; Preferably, the product is a topical composition; Preferably, the topical composition is a pharmaceutical composition or a cosmetic composition; Optionally, the cosmetic composition further comprises a carrier, an active ingredient, an excipient, or any combination thereof; Preferably, the medium includes a diluent, a dispersant, a carrier, or any combination thereof; Preferably, the active ingredients include emollients, moisturizers, whitening active ingredients, anti-aging active ingredients, or any combination thereof; Preferably, the excipients include emulsifiers, thickeners, preservatives, fragrances, or any combination thereof; Preferably, the product is a medical aesthetic product.

9. The use of the fungal fermentation product of claim 1 or the fungal fermentation product prepared by any one of claims 2 to 7 for the preparation of products that slow down skin aging; Preferably, the slowing down of skin aging includes reducing wrinkles and / or firming the skin; Preferably, the reduction of wrinkles includes reducing the area, volume, and / or number of wrinkles, or lowering the grade of wrinkles; Preferably, the wrinkles include one or more of the following: forehead wrinkles, crow's feet wrinkles, under-eye wrinkles, and nasolabial folds.

10. The use of cyclic dipeptides or their salts in the preparation of products that slow down skin aging, wherein, The cyclic dipeptide is cyclic proline-leucine, or the cyclic dipeptide includes 2, 3, 4, 5 or 6 of the following: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine. Preferably, the slowing down of skin aging includes reducing wrinkles and / or firming the skin; Preferably, the reduction of wrinkles includes reducing the area, volume, and / or number of wrinkles, or lowering the grade of wrinkles; Preferably, the wrinkles include one or more of the following: forehead wrinkles, crow's feet wrinkles, under-eye wrinkles, and nasolabial folds.

11. Use of the fungal fermentation product of claim 1 or the fungal fermentation product prepared by any one of claims 2 to 7 for the preparation of a product, said product being used to reduce the activity of SA-β-gal in cells.

12. Use of the cyclic dipeptide or its salt for the preparation of a product for reducing the activity of SA-β-gal in cells; wherein the cyclic dipeptide is cyclic isoleucine-proline, cyclic proline-leucine, or cyclic leucine-leucine, or the cyclic dipeptide comprises 2, 3, 4, 5, or 6 of the following: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

13. The use according to any one of claims 9-12, wherein, The product is a topical skin composition; Preferably, the topical composition is a pharmaceutical composition or a cosmetic composition; Preferably, the product is a medical aesthetic product.

14. A method for reducing SA-β-Gal activity in cells, comprising administering an effective amount of a cyclic dipeptide to the cells; said cyclic dipeptide is cyclic isoleucine-proline, cyclic proline-leucine, or cyclic leucine-leucine, or said cyclic dipeptide comprises 2, 3, 4, 5, or 6 of the following: cyclic proline-tyrosine, cyclic isoleucine-proline, cyclic proline-leucine, cyclic proline-phenylalanine, cyclic valine-phenylalanine, and cyclic leucine-leucine.

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