Preparation method of dendrobium polysaccharide and cosmetic application thereof

Abstract

The application discloses a preparation method of dendrobium polysaccharide and cosmetic application thereof, and relates to the technical field of daily necessities. The preparation method of the dendrobium polysaccharide comprises the following steps: mixing dendrobium officinale powder and water to obtain a to-be-extracted solution; performing ultrasonic extraction on the to-be-extracted solution; after the ultrasonic extraction, adding a composite enzyme into the to-be-extracted solution to perform enzymolysis; and after the enzymolysis, filtering the enzymolysis solution by using an ultrafiltration membrane with a molecular weight cut-off of 5000-10000 Da. By means of the mode of 'ultrasonic extraction+enzymolysis+ultrafiltration membrane filtration', the extraction effect of the dendrobium polysaccharide can be remarkably improved, and the content of the dendrobium polysaccharide is improved; the dendrobium polysaccharide prepared has a high content and a small molecular weight. By selecting the dendrobium polysaccharide with ultra-low molecular weight (the molecular weight is usually less than 10 kDa) as the component of an antioxidant anti-wrinkle composition, the transdermal absorption efficiency and bioavailability of the antioxidant anti-wrinkle composition can be remarkably improved. The antioxidant anti-wrinkle composition can be widely applied to the preparation of skin care products or cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of daily necessities, in particular to a preparation method of dendrobium polysaccharide and cosmetic application thereof. BACKGROUND

[0002] Skin, as the largest organ of the human body, its health status directly affects the appearance and physiological function. With the influence of factors such as age growth, environmental pressure (such as ultraviolet radiation, air pollution) and living habits (such as staying up late, unbalanced diet), skin will appear problems such as oxidative stress damage and wrinkle formation. Oxidative stress will lead to excessive production of free radicals, damage to lipids, proteins and DNA in skin cells, and accelerate skin aging; and the degradation of collagen and elastic fibers is one of the main reasons for wrinkle formation.

[0003] Dendrobium officinale contains active ingredients such as polysaccharides, flavonoids, alkaloids, amino acids and trace elements. These components show potential effects such as moisturizing, antioxidant, soothing and repairing in vitro experiments or industry research, so they are widely used in skin care products (such as cream, essence, mask), makeup (such as primer) and other categories, and become a popular choice in natural plant skin care raw materials.

[0004] However, the current extraction efficiency of Dendrobium officinale is low, and the polysaccharide content of Dendrobium officinale obtained is low, which leads to poor effect.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application relates to the technical field of daily necessities, in particular to a preparation method of dendrobium polysaccharide and cosmetic application thereof.

[0007] The present application is realized as follows:

[0008] In a first aspect, the present application provides a preparation method of dendrobium polysaccharide, which comprises:

[0009] Mixing Dendrobium officinale powder with water to obtain a to-be-extracted solution;

[0010] Ultrasonic extraction is performed on the to-be-extracted solution, which comprises extracting for 20-40 min at a first power, then continuing to extract for 10-20 min at a second power, and then continuing to extract for 10-30 min at a third power; wherein the first power is 180-220 W, and the second power and the third power are different, wherein the second power is 280-320 W and the third power is 380-420 W; or the second power is 380-420 W and the third power is 280-320 W;

[0011] After ultrasonic extraction, a complex enzyme is added to the to-be-extracted liquid for enzymolysis, the complex enzyme including cellulase, pectinase and alkaline protease in a mass ratio of 5-9:1-5:3-7;

[0012] After the enzymolysis is completed, the enzymolysis liquid is filtered by using an ultrafiltration membrane with a molecular weight cut-off of 5000-10000 Da.

[0013] In an optional embodiment, the solid-liquid ratio of the Dendrobium candidum powder to the water is 1g:10-20mL;

[0014] And / or, the extraction temperature of the ultrasonic extraction is 45-60℃;

[0015] And / or, the pH of the system during the ultrasonic extraction is 5.5-7.

[0016] In an optional embodiment, the added amount of the complex enzyme is 0.9-2.1% of the mass of the Dendrobium candidum powder;

[0017] And / or, the temperature of the enzymolysis is 50-60℃, and the enzymolysis time is 2-5h;

[0018] And / or, the pH of the system during the enzymolysis is 4.8-6;

[0019] In an optional embodiment, the ultrafiltration membrane is cyclically ultrafiltrated under a pressure of 0.08-0.12MPa.

[0020] In an optional embodiment, the preparation method of the Dendrobium candidum powder includes: after the fresh Dendrobium candidum stem segments are freeze-dried, the freeze-dried Dendrobium candidum stem segments are crushed through an 80-100 mesh sieve.

[0021] In an optional embodiment, the temperature of the freeze-drying is-50℃~-30℃, and the time of the freeze-drying is 20h~30h.

[0022] In a second aspect, the present application provides a Dendrobium polysaccharide, which is prepared by using the preparation method of the Dendrobium polysaccharide according to any one of the preceding embodiments.

[0023] In a third aspect, the present application provides an anti-oxidation and anti-wrinkle composition, which includes, in terms of mass percentage, 60%-85% of a Dendrobium polysaccharide, 15%-25% of ergothioneine and 5%-15% of a decarboxylated dipeptide hydrochloride, and the Dendrobium polysaccharide is prepared by using the preparation method of the Dendrobium polysaccharide according to the preceding embodiments.

[0024] In a fourth aspect, the present application provides a use of the anti-oxidation and anti-wrinkle composition according to the preceding embodiments in the preparation of a skin care product or a cosmetic product, and the anti-oxidation and anti-wrinkle composition is added in an amount of 0.01-5wt% in the skin care product or the cosmetic product.

[0025] In an optional embodiment, the skin care product or cosmetic product comprises at least one of skin care water, skin care emulsion, skin care essence, skin care cream, whitening cream, anti-wrinkle cream, anti-aging cream, sunscreen cream, BB cream, foundation cream, and facial mask.

[0026] The present application has the following beneficial effects:

[0027] The preparation method of the dendrobium polysaccharide provided by the present application can significantly improve the extraction effect of the dendrobium polysaccharide, increase the content of the dendrobium polysaccharide, and obtain the dendrobium polysaccharide with high content and small molecular weight. By selecting the dendrobium polysaccharide with ultra-low molecular weight (molecular weight is usually less than 10 kDa) as the component of the anti-oxidation and anti-wrinkle composition, the transdermal absorption efficiency and bioavailability of the dendrobium polysaccharide can be significantly improved compared with other dendrobium polysaccharides. Moreover, the dendrobium polysaccharide, ergothioneine and decarboxyed peptide hydrochloride in the anti-oxidation and anti-wrinkle composition have a synergistic effect, which can slow down the evaporation of water in the stratum corneum, produce moisturizing and skin barrier repairing effects on the skin surface, and have a high moisturizing effect. The anti-oxidation and anti-wrinkle composition can be widely used in the preparation of skin care products or cosmetic products, and has safety, mildness and fast effect. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0029] Figure 1 The yield of the dendrobium polysaccharide of examples 1.1~1.8 and comparative examples 1.1~1.8 in experimental example one is shown in the diagram.

[0030] Figure 2 The content of hyaluronic acid of examples 2.1~2.6 and comparative examples 2.1~2.6 in experimental example two is shown in the diagram. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer can be obtained by market purchase.

[0032] The application provides a preparation method of dendrobium polysaccharide, which comprises the following steps: mixing dendrobium officinale powder with water to obtain a to-be-extracted solution; performing ultrasonic extraction on the to-be-extracted solution; after the ultrasonic extraction, adding a composite enzyme into the to-be-extracted solution for enzymolysis; and filtering the enzymolysis solution by using an ultrafiltration membrane after the enzymolysis.

[0033] In the application, the method of "ultrasonic extraction + enzymolysis + ultrafiltration membrane filtration" can significantly improve the extraction effect of dendrobium polysaccharide and improve the content of dendrobium polysaccharide.

[0034] The preparation method of the dendrobium officinale powder comprises the following steps: freezing and drying fresh dendrobium officinale stem segments, the freezing and drying temperature is-50℃ to-30℃, and the freezing and drying time is 20h to 30h, and then crushing and sieving through an 80-100 mesh sieve.

[0035] The ultrasonic extraction comprises the following steps: extracting for 20-40min under a first power, then continuing to extract for 10-20min under a second power, and then continuing to extract for 10-30min under a third power; the first power is 180-220W, the second power is different from the third power, the second power is 280-320W and the third power is 380-420W, or the second power is 380-420W and the third power is 280-320W; under the action of ultrasonic waves, liquid molecules form cavitation bubbles, the cavitation bubbles are generated and disappear at a very fast speed and a very high frequency, the cavitation bubbles release a huge amount of energy in an instant, the impact and shearing effect caused by the cavitation bubbles is great, the cell walls of the plant can be broken, and the effective components in the cells can be dissolved in the solvent faster and more.

[0036] In the ultrasonic extraction, the solid-liquid ratio of the dendrobium officinale powder and water is 1g:10-20mL, the extraction temperature is 45-60℃, and the pH of the system in the ultrasonic extraction is 5.5-7.

[0037] By controlling the parameters of the ultrasonic extraction, the cell walls of the dendrobium officinale are damaged, and the effective components in the cells are dissolved in the solvent, so that the ultrasonic extraction effect can be improved.

[0038] In the present application, the complex enzyme is used for enzymolysis, and the complex enzyme can form a multi-enzyme synergistic system by compounding multiple enzymes, and the multiple enzymes work together to break down the structural barriers of the substrate, accelerate the overall reaction, and solve the problem of single enzyme in complex substrate, which is not comprehensive and low efficiency. Specifically, the complex enzyme in the present application includes cellulase, pectinase and alkaline protease in a mass ratio of 5-9:1-5:3-7; the addition amount of the complex enzyme is 0.9-2.1% of the mass of the dendrobium officinale powder; the temperature of the enzymolysis is 50-60℃, and the enzymolysis time is 2-5h; and the pH of the system during the enzymolysis is 4.8-6.

[0039] After the enzymolysis, the present application also uses an ultrafiltration membrane for filtration, and the molecular weight cut-off of the ultrafiltration membrane is 5000-10000Da; and the ultrafiltration membrane is subjected to cyclic ultrafiltration under a pressure of 0.08-0.12MPa. The "cyclic ultrafiltration" refers to that the feed liquid flows in the membrane system in a cycle, and the feed liquid does not pass through the membrane assembly once, but forms a circulating flow in the membrane system by the action of a pump (part of the feed liquid passes through the membrane to become "permeate" containing small molecules; the "retentate" that does not pass through the membrane is returned to the feed liquid pool to continue to participate in the circulation). This circulation mechanism can improve the flow rate of the feed liquid on the membrane surface, reduce the membrane pollution (such as solute deposition to block the membrane pores), and at the same time, make the feed liquid fully contact with the membrane, and improve the separation efficiency.

[0040] By the above-mentioned "ultrasonic extraction + enzymolysis + ultrafiltration membrane filtration" mode, the extraction effect of the dendrobium officinale polysaccharide can be significantly improved, and the content of the dendrobium officinale polysaccharide is improved.

[0041] Further, the present application also provides an anti-oxidation and anti-wrinkle composition, and the raw materials of the anti-oxidation and anti-wrinkle composition include dendrobium officinale polysaccharide 60%-85%, ergothioneine 15%-25% and decarboxy peptides hydrochloride 5%-15% by mass percentage, and the dendrobium officinale polysaccharide is the dendrobium officinale polysaccharide as described in the foregoing embodiments.

[0042] Preferably, the raw materials of the anti-oxidation and anti-wrinkle composition include dendrobium officinale polysaccharide 77%, ergothioneine 17% and decarboxy peptides hydrochloride 6% by mass percentage.

[0043] At present, the anti-oxidation and anti-wrinkle products on the market mostly use single component or simple compounding, and there are problems such as single target point, limited effect, poor transdermal absorption and the like. The anti-oxidation and anti-wrinkle composition provided by the present application uses dendrobium officinale polysaccharide, ergothioneine and decarboxy peptides hydrochloride for compounding, and the three components play a synergistic effect, can efficiently penetrate, and have important market demand and application value.

[0044] The super low molecular weight (usually less than 10 kDa) dendrocalamus latiflorus polysaccharide is selected as the component of the anti-oxidation and anti-wrinkle composition in the present application, which can significantly improve the transdermal absorption efficiency and bioavailability compared with other dendrocalamus latiflorus polysaccharides. The dendrocalamus latiflorus polysaccharide contains a large number of hydrophilic hydroxyl groups, which can form a biological film on the skin surface, slow down the evaporation of water in the stratum corneum, and produce effects such as moisturizing and repairing the skin barrier on the skin surface. The dendrocalamus latiflorus polysaccharide can act on the main molecular basis clock protein (CLOCK) of the biological clock and brain and muscle aromatic hydrocarbon receptor nuclear transport-like protein 1 (BMAL1), thereby regulating the rhythm of skin disorders, promoting the expression of epidermal aquaporin 3 (AQP3), enhancing the moisturizing performance of the skin, and improving the problems of skin water loss and peeling caused by circadian rhythm disorders.

[0045] Ergothioneine is a natural antioxidant with unique cell protection effect, which can scavenge various free radicals and participate in the intracellular antioxidant defense system. Ergothioneine can efficiently scavenge free radicals in the body, such as hydroxyl radicals and hydrogen peroxide, and has a significant antioxidant capacity, which is 3 times that of adibend, 14 times that of glutathione, and 30 times that of coenzyme Q10. It can effectively protect mitochondria from free radical damage, thereby protecting DNA from damage and maintaining the normal function and vitality of cells.

[0046] Decarboxylated carnosine hydrochloride is a highly efficient antioxidant that can inhibit protein glycosylation and lipid peroxidation. Compared with carnosine, decarboxylated carnosine hydrochloride has better stability. The molecular model method was used to investigate that decarboxylated carnosine has better transdermal ability and stability than carnosine. There is carnosinase in the human body that can decompose carnosine, that is, in the skin "food chain", carnosine is easily eaten by its natural enemy "carnosinase", but decarboxylated carnosine does not have this problem because it cannot be recognized and decomposed by carnosinase. Decarboxylated carnosine hydrochloride can convert harmful peroxidized lipids (LOOH) into non-toxic alcohols (LOH), thereby protecting cells from oxidative damage, reducing the damage of free radicals to the skin, and delaying skin aging. The imidazole group on the structure of decarboxylated carnosine hydrochloride enables it to have strong metal ion chelating ability, which can prevent excessive metal ions (such as Cu 2+ , Fe 2+ , etc.) from catalyzing free radicals, further enhancing antioxidant capacity.

[0047] The preparation method of the anti-oxidation and anti-wrinkle composition is simple, which only needs to mix the above components uniformly. Specifically, decarboxylated carnosine hydrochloride, dendrocalamus latiflorus polysaccharide and ergothioneine are added into a beaker in sequence, and stirred while adding, until mixed uniformly.

[0048] The anti-oxidation and anti-wrinkle composition can be widely used in the preparation of skin care products or cosmetics. In the application of skin care products, the anti-oxidation and anti-wrinkle composition is safe and mild, and has a rapid effect. The addition amount of the anti-oxidation and anti-wrinkle composition in the skin care product or cosmetic is 0.01-5wt%. The skin care product or cosmetic includes but is not limited to at least one of skin care water, skin care emulsion, skin care essence, skin care cream, whitening cream, anti-wrinkle cream, anti-aging cream, sunscreen cream, BB cream, sunscreen cream, and facial mask.

[0049] The features and performances of the present application are further described in detail in the following combined with examples.

[0050] Example 1.1

[0051] The present example provides a dendrobium polysaccharide, and a preparation method thereof includes: selecting fresh dendrobium candidum stems, washing and cutting into segments, grinding through an 80-mesh sieve after freeze-drying (-40℃, 24h), to obtain standardized dendrobium candidum powder. The solid-liquid ratio is 1:20 (g / mL), pH=7.0, 60℃, 200W ultrasonic extraction for 20min, 400W ultrasonic extraction for 10min, and 300W ultrasonic extraction for 30min. Then, composite enzymes are used for enzymolysis at 50℃ and pH=6.0 for 5h, the composite enzymes including 0.7% cellulase, 0.3% pectinase, and 0.5% alkaline protease (the addition amount of the composite enzymes is 1.5% of the mass of the dendrobium candidum powder), and after the enzymolysis is completed, 10000Da ultrafiltration membrane and 0.08MPa pressure are used for cyclic ultrafiltration.

[0052] Example 1.2

[0053] The present example is basically the same as example 1, and the difference is that the ultrasonic extraction method of the present comparative example is different. In the present example, 200W ultrasonic extraction is performed for 30min, 400W ultrasonic extraction is performed for 10min, and 300W ultrasonic extraction is performed for 20min.

[0054] Example 1.3

[0055] The present example is basically the same as example 1, and the difference is that the ultrasonic extraction method of the present comparative example is different. In the present example, 200W ultrasonic extraction is performed for 30min, 400W ultrasonic extraction is performed for 10min, and 300W ultrasonic extraction is performed for 20min.

[0056] Example 1.4

[0057] The present example is basically the same as example 1, and the difference is that the ultrasonic extraction method of the present comparative example is different. In the present example, 200W ultrasonic extraction is performed for 30min, 400W ultrasonic extraction is performed for 10min, and 300W ultrasonic extraction is performed for 20min.

[0058] Example 1.5

[0059] The present example is basically the same as example 1, the difference is only that the ultrasonic extraction method of the present example is different, in the present example, 200W ultrasonic extraction for 40min, 300W ultrasonic extraction for 10min, 400W ultrasonic extraction for 10min.

[0060] Example 1.6

[0061] The present example provides a dendrobium polysaccharide, and the preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40℃, 24h), crushing and passing through an 80 mesh sieve to obtain standardized dendrobium candidum powder. The solid-liquid ratio is 1:15 (g / mL), pH=6.0, 50℃, 180W ultrasonic extraction for 40min, 380W ultrasonic extraction for 20min, and 280W ultrasonic extraction for 30min. Then, under the conditions of 55℃ and pH=5.0, the compound enzyme is used for enzymolysis for 3h, the compound enzyme comprises 0.7% cellulase, 0.3% pectinase and 0.5% alkaline protease (the compound enzyme is added in an amount of 1.5% of the mass of the dendrobium candidum powder), and after the enzymolysis is completed, 7000Da ultrafiltration membrane and 0.1MPa pressure are used for cyclic ultrafiltration.

[0062] Example 1.7

[0063] The present example provides a dendrobium polysaccharide, and the preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40℃, 24h), crushing and passing through an 80 mesh sieve to obtain standardized dendrobium candidum powder. The solid-liquid ratio is 1:12 (g / mL), pH=5.5, 45℃, 220W ultrasonic extraction for 10min, 420W ultrasonic extraction for 10min, and 320W ultrasonic extraction for 10min. Then, under the conditions of 60℃ and pH=4.8, the compound enzyme is used for enzymolysis for 2.5h, the compound enzyme comprises 0.7% cellulase, 0.3% pectinase and 0.5% alkaline protease (the compound enzyme is added in an amount of 1.5% of the mass of the dendrobium candidum powder), and after the enzymolysis is completed, 5000Da ultrafiltration membrane and 0.12MPa pressure are used for cyclic ultrafiltration.

[0064] Example 1.8

[0065] The embodiment provides a dendrobium polysaccharide, and a preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40 DEG C, 24h), crushing and passing through an 80-mesh screen to obtain standardized dendrobium candidum powder. The material-liquid ratio is 1:20 (g / mL), pH=7.0, 60 DEG C, 200W ultrasonic extraction for 20min, 400W ultrasonic extraction for 10min, and 300W ultrasonic extraction for 30min. Then, composite enzymes are used for enzymolysis under the conditions of 50 DEG C and pH=5.2 for 4h, the composite enzymes include 0.7% cellulase, 0.3% pectinase and 0.5% alkaline protease (the composite enzyme addition amount is 1.5% of the mass of the dendrobium candidum powder), and after the enzymolysis, 10000Da ultrafiltration membrane and 0.08MPa pressure are used for cyclic ultrafiltration.

[0066] Comparative example 1.1: traditional hot water extraction method

[0067] The comparative example provides a dendrobium polysaccharide, and a preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40 DEG C, 24h), crushing and passing through an 80-mesh screen to obtain standardized dendrobium candidum powder.

[0068] The extraction conditions are as follows: the material-liquid ratio is 1:20 (g / mL), 90 DEG C hot water immersion extraction is carried out for 2h, and the operation is repeated for three times.

[0069] The purification steps are as follows: the water extraction liquid is precipitated by 4 times of ethanol, the precipitate is collected by centrifugation (3000rpm, 15min), and freezing and drying are carried out.

[0070] The characteristics are as follows: the polysaccharide molecular weight distribution is wide (10-500kDa), and additional enzymolysis treatment is needed.

[0071] Comparative example 1.2: single enzymolysis extraction method

[0072] The comparative example provides a dendrobium polysaccharide, and a preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40 DEG C, 24h), crushing and passing through an 80-mesh screen to obtain standardized dendrobium candidum powder.

[0073] The extraction conditions are as follows: the material-liquid ratio is 1:15 (g / mL), 0.7% cellulase is directly added, and enzymolysis is carried out at 55 DEG C for 5h.

[0074] The purification steps are as follows: the enzymolysis liquid is directly spray-dried after enzyme inactivation and centrifugation.

[0075] Comparative example 1.3: single enzymolysis extraction method

[0076] The comparative example provides a dendrobium polysaccharide, and a preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40 DEG C, 24h), crushing and passing through an 80-mesh screen to obtain standardized dendrobium candidum powder.

[0077] Extraction condition: solid-liquid ratio 1:15 (g / mL), directly adding 0.3% pectinase, enzymolysis for 5h at 55℃.

[0078] Purification step: after enzyme inactivation and centrifugation, the enzymolysis solution was directly spray-dried.

[0079] Comparative Example 1.4: single enzymolysis extraction method

[0080] This comparative example provides a dendrobium polysaccharide, and the preparation method thereof comprises the following steps: selecting fresh dendrobium candidum stems, washing and cutting, freezing and drying (-40℃, 24h), crushing through an 80-mesh sieve, and obtaining standardized dendrobium candidum powder.

[0081] Extraction condition: solid-liquid ratio 1:15 (g / mL), directly adding 0.5% alkaline protease, enzymolysis for 5h at 55℃.

[0082] Purification step: after enzyme inactivation and centrifugation, the enzymolysis solution was directly spray-dried.

[0083] Comparative Example 1.5

[0084] This comparative example is basically the same as Example 1.1, and the only difference is that in this comparative example, the ultrasonic extraction condition is: ultrasonic extraction at 200W for 40min and ultrasonic extraction at 400W for 20min.

[0085] Comparative Example 1.6

[0086] This comparative example is basically the same as Example 1.1, and the only difference is that in this comparative example, the ultrasonic extraction condition is: ultrasonic extraction at 200W for 40min and ultrasonic extraction at 300W for 20min.

[0087] Comparative Example 1.7

[0088] This comparative example is basically the same as Example 1.1, and the only difference is that in this comparative example, the ultrasonic extraction condition is: ultrasonic extraction at 200W for 60min.

[0089] Comparative Example 1.8

[0090] This comparative example is basically the same as Example 1.1, and the only difference is that in this comparative example, after the enzymolysis is completed, the 10000Da ultrafiltration membrane is not used for 0.08MPa pressure circulation ultrafiltration, and the filtration is directly performed.

[0091] Experimental Example One

[0092] Examples 1.1-1.8 and Comparative Examples 1.1-1.8, while adding pH 4 sodium phosphate dibasic-citric acid buffer, shake well, react in a 50°C constant temperature water bath for 90 min. Immediately after the end of the reaction, inactivate the complex enzyme in boiling water, filter with a suction filter bottle, take 1 mL of the filtrate, and measure the absorbance A value at 490 nm. Calculate the yield of dendrobium polysaccharide, and the calculation results are shown in Table 1. Figure 1 .

[0093] From Figure 1 It can be seen that the yield of dendrobium polysaccharide prepared by the method of Example 1.6 is the highest, and the yield of dendrobium polysaccharide obtained by Comparative Examples 1.1-1.8 is significantly lower than the yield of dendrobium polysaccharide of Examples 1.1-1.8.

[0094] The dendrobium polysaccharide prepared by the above Example 1.1 is used as a raw material to prepare the anti-oxidation and anti-wrinkle compositions provided in Examples 2.1-2.6 and Comparative Examples 2.1-2.6.

[0095] Example 2.1

[0096] The anti-oxidation and anti-wrinkle composition provided in this example includes, by mass percentage, 60% of dendrobium polysaccharide, 25% of ergothioneine, and 15% of decarboxylated carnosine hydrochloride, and the preparation method includes stirring and mixing the above raw materials uniformly at room temperature.

[0097] Example 2.2

[0098] The anti-oxidation and anti-wrinkle composition provided in this example includes, by mass percentage, 74% of dendrobium polysaccharide, 14% of ergothioneine, and 12% of decarboxylated carnosine hydrochloride, and the preparation method includes stirring and mixing the above raw materials uniformly at room temperature.

[0099] Example 2.3

[0100] The anti-oxidation and anti-wrinkle composition provided in this example includes, by mass percentage, 75% of dendrobium polysaccharide, 18% of ergothioneine, and 7% of decarboxylated carnosine hydrochloride, and the preparation method includes stirring and mixing the above raw materials uniformly at room temperature.

[0101] Example 2.4

[0102] The anti-oxidation and anti-wrinkle composition provided in this example includes, by mass percentage, 70% of dendrobium polysaccharide, 23% of ergothioneine, and 7% of decarboxylated carnosine hydrochloride, and the preparation method includes stirring and mixing the above raw materials uniformly at room temperature.

[0103] Example 2.5

[0104] The embodiment provides an anti-oxidation and anti-wrinkle composition, raw materials of which include dendrobium polysaccharide 72%, ergothioneine 18% and de-carboxy myo-inositol peptide hydrochloride 10% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0105] Example 2.6

[0106] The embodiment provides an anti-oxidation and anti-wrinkle composition, raw materials of which include dendrobium polysaccharide 65%, ergothioneine 24% and de-carboxy myo-inositol peptide hydrochloride 11% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0107] Comparative Example 2.1

[0108] The comparative example provides an anti-oxidation and anti-wrinkle composition, raw materials of which include dendrobium polysaccharide 75% and ergothioneine 25% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0109] Comparative Example 2.2

[0110] The comparative example provides an anti-oxidation and anti-wrinkle composition, raw materials of which include dendrobium polysaccharide 85% and de-carboxy myo-inositol peptide hydrochloride 15% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0111] Comparative Example 2.3

[0112] The embodiment provides an anti-oxidation and anti-wrinkle composition, raw materials of which include ergothioneine 50% and de-carboxy myo-inositol peptide hydrochloride 50% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0113] Comparative Example 2.4

[0114] The embodiment provides an anti-oxidation and anti-wrinkle composition, raw materials of which include dendrobium polysaccharide 75%, ergothioneine 18% and purified water 7% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0115] Comparative Example 2.5

[0116] The embodiment provides an anti-oxidation and anti-wrinkle composition, raw materials of which include dendrobium polysaccharide 75%, purified water 18% and de-carboxy myo-inositol peptide hydrochloride 7% by mass percentage, and a preparation method thereof includes stirring and uniformly mixing the raw materials at room temperature.

[0117] Comparative Example 2.6

[0118] This embodiment provides an antioxidant and anti-wrinkle composition, the raw materials of which, by mass percentage, include 75% purified water, 18% ergothioneine and 7% decarboxylated carnosine hydrochloride. The preparation method includes stirring and mixing the above raw materials evenly at room temperature to obtain the composition.

[0119] Examples 2.1 to 2.6 and Comparative Examples 2.1 to 2.6 provide an antioxidant and anti-wrinkle composition, the raw materials of which are shown in Table 1.

[0120] Table 1. Raw material statistics for different examples

[0121]

[0122] Experimental Example 2

[0123] Experiments were conducted on Examples 2.1 to 2.6 and Comparative Examples 2.1 to 2.6 above, and the hyaluronic acid content was measured.

[0124] The experimental method included: HSF cells in the logarithmic growth phase were seeded into culture flasks, and when the density exceeded 80%, they were digested with trypsin and collected for counting. The cell suspension was diluted with complete culture medium (90% H-DMEM + 9% FBS + 1% PS) (v / v) to a cell count of 1.0 × 10⁻⁶ cells. 5 Cells were seeded in 6-well plates. Incubated at 37°C with 5% CO2 for 6–12 hours to allow for normal cell spread. The complete culture medium was then discarded, and the cells were divided into a blank control group, a positive control group, and an experimental group. Corresponding conditioned medium was added to each group, and cells and supernatant were collected after 24 hours of incubation. Experimental groups: Complete culture medium was used for different example groups and comparative groups.

[0125] Cell supernatants were collected, and HA expression levels in the samples were determined using the corresponding ELISA kits according to the manufacturer's instructions using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Microplates were coated with purified hyaluronic acid-binding protein (HABP) to prepare a solid-phase antibody. HA, biotinylated anti-rat hyaluronic acid-binding protein (HABP), and horseradish peroxidase (HRP)-labeled avidin were added sequentially to the wells coated with the monoclonal antibody. After thorough washing, the samples were developed using the substrate 3,3′,5,5′-tetramethylbenzidine (TMB). TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the hyaluronic acid content in the sample. The absorbance (OD value) was measured at 450 nm using an ELISA reader, and the sample concentration was calculated.

[0126] Please refer to the test results. Figure 2 .

[0127] from Figure 2It can be seen that the anti-oxidation and anti-wrinkle composition provided by the embodiment of the present application has a higher hyaluronic acid content, and the comparative example lacking of certain element has a significantly reduced hyaluronic acid content.

[0128] The mechanism of action between the three components of the anti-oxidation and anti-wrinkle composition provided by the present application is as follows:

[0129] When ergothioneine, dendrobium polysaccharide and de-carboxylated myo-inositol hydrochloride synergistically act in the field of anti-oxidation and anti-wrinkle, they can construct a three-dimensional defense-repair network covering multiple pathways of skin aging, achieving an effect far beyond that of a single component. The following are the specific mechanisms and advantages of the synergistic action of the three: skin aging is mainly driven by multiple factors such as oxidative stress, glycation, inflammatory damage, collagen degradation, and barrier damage. The three components have clear division of labor, cover different targets, and form a complement. Ergothioneine treats exogenous oxidative damage (immediate free radical scavenging), dendrobium polysaccharide activates the endogenous antioxidant system and barrier repair / moisturizing, and de-carboxylated myo-inositol hydrochloride resists glycation damage (protects collagen structure). The synergistic action of the three components plays a role in multiple targets such as free radical scavenging, inhibition of oxidative stress, promotion of collagen synthesis, and repair of skin barrier, and the effect is significantly improved compared with a single component.

[0130] Experimental Example Three

[0131] The anti-oxidation and anti-wrinkle composition provided in the above embodiment 2.5 is added to a face cream to prepare a face cream containing 0.2% of the anti-oxidation and anti-wrinkle composition. The formula ingredient table of the face cream is as follows:

[0132] Table 2. Formula ingredient table of the face cream

[0133]

[0134] The preparation method of the face cream is as follows: the isohexyl alcohol, dimethicone, caprylic / capric triglyceride, tri (laureth-4) phosphate and glyceryl stearate in phase B are sequentially added to a clean oil phase kettle, heated to 80℃, and stirred to dissolve uniformly, and kept for 10 min; the purified water, acryloyldimethyltaurine ammonium / VP copolymer, butanediol, carbomer and xanthan gum in phase A are sequentially added to a clean water phase kettle, homogenized at high speed for 3 min, then heated to 80℃, kept for 15 min, and fully stirred to dissolve completely; phase A and phase B are extracted into an emulsification kettle, homogenized at high speed for 5 min, then continuously stirred for 8 min; the phenoxyethanol, hexanediol and ethylhexylglycerin in phase C are sequentially added to the kettle while cooling water is used to cool to 45℃; finally, the anti-oxidation and anti-wrinkle composition in phase D is added; after fully stirring uniformly, the temperature is reduced to 38℃, the material is discharged and filtered, and after passing the inspection, the face cream sample is obtained.

[0135] According to the prepared face cream sample, the anti-wrinkle efficacy evaluation before and after product use is carried out.

[0136] Volunteers: Chinese healthy female subjects aged 30 to 55 years;

[0137] After cleansing the face in the morning and evening, take an appropriate amount of the product and evenly apply it to the facial skin, gently massage until completely absorbed. Use it 2 times a day, once in the morning and once in the evening, for 2 consecutive weeks. During the test period, the subjects are prohibited from using soothing, firming, and anti-wrinkle preparations; the subjects are prohibited from applying, injecting, orally taking, or otherwise ingesting preparations that affect the soothing, firming, and anti-wrinkle test in other forms: the subjects mainly engage in indoor activities, avoiding long-term exposure to outdoor light.

[0138] After facial cleansing for 30 minutes, use VISIA7 facial skin imaging analyzer to collect facial images and analyze the periorbital wrinkle count; use PRIMOS-CR to collect wrinkle images of the facial measurement area of the subject, determine the target analysis position of the wrinkles, and use the analysis software to obtain the volume and number of under-eye wrinkles and marionette wrinkles; and use CM825, GL200, CL400, and MX18 to detect the water content of the stratum corneum, skin gloss, skin brightness L* value, and skin hemoglobin content at the test site; the measurement values are taken as the baseline values (D0). Then the product is distributed and on-site use is guided. The subjects need to use CM825 to detect the same indicators 15 minutes after using the product (DOT15min); and revisit and detect the same indicators except CM825 7 days and 14 days after using the product (D7 and D14).

[0139] The same instrument and the same tester are used to complete the test of the same subject. The test site should be kept consistent before and after between the two test time points.

[0140] Wrinkle count analysis

[0141] The number of periorbital wrinkles can be obtained by software analysis of the standard light image taken by VISIA7. By comparing the change in the number of periorbital wrinkles before and after use, the anti-wrinkle efficacy of the product can be reflected.

[0142] The greater the decrease in the number of periorbital wrinkles at different revisit time points compared with the baseline value (D0) before use, the better the improvement effect of periorbital wrinkles, indicating that the anti-wrinkle effect of the product is better.

[0143] Wrinkle number change rate = (wrinkle number after using the product - wrinkle number before using the product) / wrinkle number before using the product x 100%.

[0144] Table 3. Descriptive statistics of periorbital wrinkle count

[0145]

[0146] Table 4. Difference statistics of periorbital wrinkle count

[0147]

[0148] The periorbital wrinkle count showed a decreasing trend after product use, decreasing by 10.17% and 10.34% after 7 days and 14 days (D7 and D14) of product use, respectively, both of which were significantly different from the baseline value (p<0.01). The periorbital wrinkle count after 7 days and 14 days of product use was lower than the baseline value.

[0149] The wrinkle number value represents the number of wrinkles in the analysis area. The lower the index value, the fewer the number of wrinkles. By comparing the change in wrinkle number value before and after use, the anti-wrinkle efficacy of the product can be reflected.

[0150] The more the number of under-eye wrinkles and marionette lines decreases at different follow-up time points compared to the baseline value (D0) before use, the better the anti-wrinkle effect of the product.

[0151] Wrinkle number change rate = (wrinkle number after product use - wrinkle number before product use) / wrinkle number before product use x 100%.

[0152] Table 5. Statistical table of wrinkle number difference

[0153]

[0154] Under-eye wrinkles: Compared with the baseline value (D0) before use, the number of under-eye wrinkles showed a decreasing trend after product use, decreasing by 21.54% and 22.31% after 7 days and 14 days (D7 and D14) of product use, respectively, both of which were significantly different from the baseline value (p<0.01). The number of under-eye wrinkles after 7 days and 14 days of product use was lower than the baseline value.

[0155] Marionette lines: Compared with the baseline value (D0) before use, the number of marionette lines showed a decreasing trend after product use, decreasing by 24.29% and 25.60% after 7 days and 14 days (D7 and D14) of product use, respectively, both of which were significantly different from the baseline value (p<0.01). The number of marionette lines after 7 days and 14 days of product use was lower than the baseline value.

[0156] In summary, the preparation method of the dendrobium polysaccharide provided by the present application can significantly improve the extraction effect of the dendrobium polysaccharide, improve the content of the dendrobium polysaccharide, and obtain the dendrobium polysaccharide with high content and small molecular weight. By selecting the dendrobium polysaccharide with ultra-low molecular weight (molecular weight is usually less than 10 kDa) as the component of the anti-oxidation and anti-wrinkle composition, the transdermal absorption efficiency and bioavailability of the dendrobium polysaccharide can be significantly improved compared with other dendrobium polysaccharides. Moreover, the dendrobium polysaccharide, ergothioneine and decarboxy-actinin hydrochloride in the anti-oxidation and anti-wrinkle composition have a synergistic effect, can slow down the evaporation of water in the stratum corneum, produce the effects of moisturizing and repairing skin barrier on the skin surface, and have the effect of high moisturizing. The anti-oxidation and anti-wrinkle composition can be widely applied to the preparation of skin care products or cosmetics, is safe and mild, and has fast effect.

[0157] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antioxidant and anti-wrinkle composition, characterized in that, The raw materials, by mass percentage, include: 60%-85% Dendrobium polysaccharide, 15%-25% ergothioneine, and 5%-15% decarboxylated carnosine hydrochloride. The preparation method of the Dendrobium polysaccharide includes: Dendrobium officinale powder is mixed with water, wherein the solid-liquid ratio of Dendrobium officinale powder to water is 1g:10-20mL, to obtain the extract to be obtained; The extract to be extracted is subjected to ultrasonic extraction, which includes extraction at a first power for 20-40 min, followed by extraction at a second power for 10-20 min, and then extraction at a third power for 10-30 min; wherein the first power is 180-220 W, and the second power is different from the third power, wherein when the second power is 280-320 W, the third power is 380-420 W; or, when the second power is 380-420 W, the third power is 280-320 W; the extraction temperature of the ultrasonic extraction is 45-60℃; and the pH of the system during the ultrasonic extraction is 5.5-7. After ultrasonic extraction, a compound enzyme is added to the extract for enzymatic hydrolysis. The amount of the compound enzyme added is 0.9-2.1% of the mass of the Dendrobium officinale powder. The compound enzyme includes cellulase, pectinase and alkaline protease in a mass ratio of 5-9:1-5:3-7. The pH of the system during enzymatic hydrolysis is 4.8-6, the temperature of enzymatic hydrolysis is 50-60℃, and the time of enzymatic hydrolysis is 2-5 hours. After enzymatic hydrolysis, the hydrolysate is filtered using an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da. The ultrafiltration membrane is circulated under a pressure of 0.08-0.12 MPa.

2. The antioxidant and anti-wrinkle composition according to claim 1, characterized in that, The preparation method of the Dendrobium officinale powder includes: freeze-drying fresh Dendrobium officinale stem segments and then pulverizing them through an 80-100 mesh sieve.

3. The antioxidant and anti-wrinkle composition according to claim 2, characterized in that, The freeze-drying temperature is -50℃ to -30℃, and the time is 20h to 30h.

4. The application of the antioxidant and anti-wrinkle composition as described in claim 1 in the preparation of skincare or cosmetic products, characterized in that, The antioxidant and anti-wrinkle composition is added to the skin care or cosmetic product at an amount of 0.01-5 wt%.

5. The application according to claim 4, characterized in that, The skin care products or cosmetics include at least one of the following: skin care water, skin care lotion, skin care essence, skin care cream, whitening cream, anti-wrinkle cream, anti-aging cream, sunscreen, BB cream, isolation cream, and face mask.

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