Preparation method and application of camellia polysaccharide with anti-aging and repairing effects

Abstract

The present invention provides a method for preparing camellia polysaccharide with anti-aging and repairing effects from camellia and its application. The preparation method of the present invention comprises: drying the camellia after supercritical CO2 extraction and ethanol ultrasonic extraction; then subjecting the dried camellia to ultrasonic extraction and enzymatic extraction to obtain a camellia polysaccharide extract; concentrating the camellia polysaccharide extract, precipitating with alcohol, etc., to obtain a camellia crude polysaccharide; filtering the camellia crude polysaccharide with an ultrafiltration membrane to obtain a camellia polysaccharide. The camellia polysaccharide prepared by the present invention has a purity of up to 89.78% and a uronic acid content of up to 35.04%. It has the effects of moisturizing, promoting collagen synthesis and inhibiting collagen degradation, repairing, anti-oxidation, whitening, etc. The cosmetics prepared by the camellia polysaccharide have good effects of anti-wrinkle, repairing, brightening skin tone, improving skin elasticity, etc.

Description

Technical Field

[0001] The invention belongs to the field of camellia deep processing technology and application, and specifically relates to a preparation method and application of camellia polysaccharide with anti-aging and repairing effects. The camellia polysaccharide of the invention has the characteristics of high purity and rich in uronic acid, and has the effects of promoting collagen synthesis and inhibiting collagen degradation, repairing, anti-oxidation, whitening, etc., and can be widely used in cosmetics as a functional raw material. Background Art

[0002] Camellia is a plant of the Camellia genus of the Theaceae family. In addition to its ornamental and environmental beautification value, camellia also has certain medicinal value. Camellia is rich in active ingredients such as polysaccharides, proteins, polyphenols and flavonoids. Its polysaccharides show biological activities such as moisturizing, anti-oxidation, anti-aging, anti-wrinkle and repair.

[0003] Polysaccharides, also known as polysaccharides, generally refer to a class of biological macromolecules composed of 10 or more monosaccharides, which exist in higher plants, algae, fungi and animals. Acidic polysaccharides refer to polysaccharides containing acidic groups such as carboxyl or sulfate related to uronic acid, which is an important biological substance. Uronic acid is the product of the oxidation of hydroxymethyl in acidic sugar molecules to carboxyl. Modern pharmacological studies have found that natural plant acidic polysaccharides can significantly promote cell proliferation and have moisturizing, anti-wrinkle, and repair activities.

[0004] The present invention prepares high-purity camellia polysaccharide from camellia for the first time, and innovatively uses supercritical carbon dioxide and ultrasonic pretreatment to defat the camellia and remove non-sugar impurities, and adopts ethanol precipitation and ultrafiltration membrane filtration to purify and prepare camellia polysaccharide from camellia, and the prepared camellia polysaccharide has a maximum purity of up to 89.78%, and a uronic acid content of 35.04%, thereby achieving a significant improvement in anti-aging, repairing, whitening and other effects; the present invention also provides a reference for the deep processing of camellia. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of camellia polysaccharide with anti-aging and repairing effects in view of the problem of low purity of camellia polysaccharide in the prior art.

[0006] Camellia is rich in active ingredients such as polysaccharides, proteins, polyphenols and flavonoids, and its polysaccharides exhibit biological activities such as moisturizing, anti-oxidation, anti-aging, anti-wrinkle, and repair. The present invention first pre-treats camellia by supercritical carbon dioxide extraction and ultrasonic extraction, and then purifies and prepares high-purity camellia polysaccharides by ethanol precipitation and ultrafiltration membrane filtration. There is no report on the preparation method of high-purity camellia polysaccharides. The present invention can obtain high-purity camellia polysaccharides through the above method, and the obtained camellia polysaccharides can be used as anti-aging and repairing ingredients in cosmetics.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for preparing camellia polysaccharide with anti-aging and repairing effects, comprising the following steps:

[0009] A1. Crush and sieve camellia flowers, and perform supercritical CO2 extraction. After the extraction, take out the camellia powder to obtain defatted camellia powder;

[0010] A2, subjecting the defatted camellia powder to ultrasonic extraction, cooling to room temperature, filtering, and drying to obtain the camellia powder after impurities are removed;

[0011] A3, adding cellulase and water to the camellia powder after impurities are removed for ultrasonic extraction, then heating the extraction, cooling and filtering to obtain a camellia polysaccharide extract;

[0012] A4, concentrating the camellia polysaccharide extract, filtering, and obtaining a camellia polysaccharide concentrate;

[0013] A5, adding a solvent to the camellia polysaccharide concentrate to precipitate the polysaccharide, filtering, and drying the filter residue to obtain crude camellia polysaccharide;

[0014] A6. Add water to the crude camellia polysaccharide to redissolve it, perform ultrafiltration, and concentrate the filtrate by rotary evaporation to obtain camellia polysaccharide.

[0015] As an embodiment of the present invention, in step A1, the extraction pressure of supercritical CO2 extraction is 10MPa-30MPa, the extraction temperature is 35°C-60°C, and the extraction time is 30min-120min. Supercritical CO2 extraction can remove oily impurities such as lipids. The presence of lipid impurities will affect the extraction of polysaccharides and the concentration of the extract.

[0016] As an embodiment of the present invention, in step A2, the temperature of ultrasonic extraction is 40-60°C, the time is 30min-180min, and the ultrasonic power is 500W-3000W. The solvent for ultrasonic extraction is ethanol or ethanol solution, and the ethanol mass content in the ethanol solution is not less than 50% (50%-100%). The amount of solvent used is 10-30 times the mass of defatted camellia powder. Using ethanol solution for ultrasonic extraction can remove alcohol-soluble impurities and small molecule impurities, such as non-polysaccharide impurities such as flavonoids, polyphenols, saponins, oligosaccharides and oligosaccharides. Removing these impurities can not only remove impurities and improve purity, but also be more conducive to subsequent concentrated alcohol precipitation operations.

[0017] As an embodiment of the present invention, in step A3, the amount of cellulase used is 0.1-5% of the mass of the camellia powder after impurities are removed, and the amount of water used is 10-30 times the mass of the camellia powder after impurities are removed.

[0018] As an embodiment of the present invention, in step A3, the ultrasonic power of ultrasonic extraction is 500W-3000W, the ultrasonic time is 10min-60min, and the temperature of the solution during ultrasonic extraction is controlled below 40°C, preferably 20-40°C.

[0019] As an embodiment of the present invention, in step A3, the temperature-raising extraction is performed twice, the first temperature is raised to 40-70°C, and the extraction is performed for 1h-3h; the second temperature is raised to 75-95°C, and the extraction is performed for 0.5h-3h. The first temperature-raising is the optimum temperature for cellulase, which is more conducive to enzymolysis; the second temperature-raising is for better dissolution of polysaccharides. The higher the extraction temperature within a certain temperature range, the higher the polysaccharide extraction rate.

[0020] As an embodiment of the present invention, in step A4, the concentration is to concentrate the camellia polysaccharide extract to 10-30% of the original mass. If it is not concentrated, a large amount of solvent (such as ethanol, methanol, acetone) is required for precipitation, and the amount of polysaccharide precipitated is small and difficult to recover.

[0021] As an embodiment of the present invention, in step A4, filtration is performed using a 0.45 μm filter membrane. Filtration can remove non-polysaccharide substances.

[0022] As an embodiment of the present invention, in step A5, when precipitating the polysaccharide, the temperature of the camellia polysaccharide concentrate is 1-4°C, and the precipitation time is 5-12h.

[0023] As an embodiment of the present invention, in step A5, the solvent accounts for 40-80% of the volume of the total system (camellia polysaccharide concentrate and solvent). The solvent includes one or more of ethanol, methanol, and acetone. The solvent is preferably anhydrous ethanol.

[0024] As an embodiment of the present invention, in step A6, the concentration of the Camellia japonica polysaccharide in the redissolved solution is 0.5-100 mg / ml. Ultrafiltration uses an ultrafiltration membrane with a pore size of 500-10000 Da. Ultrafiltration can further remove impurities with a molecular weight of less than 500 Da, such as glycoproteins, proteins, and oligosaccharides.

[0025] As an embodiment of the present invention, in step A6, the concentrated solution obtained by rotary evaporation is dried or freeze-dried to obtain camellia polysaccharide.

[0026] The present invention also provides an application of the camellia polysaccharide in preparing cosmetics.

[0027] As an embodiment of the present invention, the cosmetics include one of lotion, essence, skin care lotion, skin care ointment, skin care cream, facial mask, and facial cleansing preparation.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention prepares high-purity camellia polysaccharide from camellia for the first time. The maximum purity of the prepared camellia polysaccharide can reach 89.78% and the uronic acid content can reach 35.04%.

[0030] (2) The present invention is unique in its process for pretreatment of camellia, and adopts a two-step impurity removal method to achieve degreasing and removal of non-sugar impurities from camellia. First, supercritical CO2 technology is used to remove lipid impurities; then, a high-concentration ethanol aqueous solution is used for ultrasonic pretreatment to remove non-sugar impurities such as saponins, polyphenols and flavonoids.

[0031] (3) The present invention adopts step-by-step extraction for enzymatic extraction of camellia polysaccharides. In the first step, enzymatic hydrolysis is performed at a suitable enzyme activity temperature, which can destroy the cell wall of the camellia and facilitate the dissolution of the polysaccharide. Then, in the second step, the polysaccharide is fully extracted into the extract by increasing the temperature.

[0032] (4) The present invention uses ethanol precipitation and ultrafiltration membrane filtration to purify the crude camellia polysaccharide, which can improve the purity of the polysaccharide and the content of uronic acid.

[0033] (5) The camellia polysaccharide prepared by the present invention has good skin care effects such as anti-wrinkle, repairing, and moisturizing, and can be used in cosmetics. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0035] The present invention relates to a method for testing the total sugar content and uronic acid content of camellia polysaccharide:

[0036] Camellia crude polysaccharide yield test method: polysaccharide yield = camellia crude polysaccharide mass (g) / camellia crude powder mass (g);

[0037] Total sugar content test method: phenol-sulfuric acid method;

[0038] Test method for uronic acid content: m-hydroxybiphenyl method;

[0039] Camellia polysaccharide purity test method: refer to Li Jianjun's academic paper "Controllable Degradation of Citrus Pectin Polysaccharide and Separation and Analysis of Oligosaccharide Fragments and Their Activity Research".

[0040] Example 1

[0041] (1) Crush and sieve camellia, and perform supercritical CO2 extraction at an extraction pressure of 10 MPa, an extraction temperature of 35° C., and an extraction time of 30 min. After the extraction is completed, take out the camellia powder to obtain defatted camellia powder;

[0042] (2) subjecting the defatted camellia powder to ultrasonic ethanol extraction to remove non-polysaccharide impurities such as flavonoids, polyphenols, and saponins. The ultrasonic extraction conditions are as follows: adding ethanol aqueous solution 10 times the mass of the defatted camellia powder, wherein the mass content of ethanol is 50%, the ultrasonic temperature is controlled at 40° C., and then ultrasonically subjected to 30 minutes of ultrasonication, wherein the ultrasonic power is 500 W;

[0043] (3) After the ultrasonic treatment is completed, the temperature of the extract is cooled to room temperature, filtered to obtain the camellia powder after impurities are removed, and then dried;

[0044] (4) adding 0.1% by weight of cellulase and 10 times of water to the defatted and impurity-removed camellia powder in step (3) for ultrasonic extraction, controlling the aqueous solution to be below 40° C., and then ultrasonicating for 10 minutes, wherein the ultrasonic power is 500 W;

[0045] (5) The temperature of the above solution was controlled at 40° C., and the solution was stirred and extracted for 1 hour; then the solution was heated to 75° C., and the solution was stirred and extracted for 0.5 hour. After cooling to room temperature, the solution was filtered to obtain a camellia polysaccharide extract;

[0046] (6) Concentrating the camellia polysaccharide extract to 10% of the original mass, filtering through a 0.45 μm filter membrane to obtain a camellia polysaccharide concentrate;

[0047] (7) adding anhydrous ethanol to the above-mentioned camellia polysaccharide concentrate to precipitate the polysaccharide, so that the volume of ethanol accounts for 40% of the total system volume, at a temperature of 1° C. and a precipitation time of 5 h;

[0048] (8) filtering and drying the precipitate from step (7) to obtain crude camellia polysaccharide;

[0049] (9) Add water to the above-mentioned crude camellia polysaccharide to redissolve it so that the concentration of the crude camellia polysaccharide is 0.5 mg / ml, and perform ultrafiltration using an ultrafiltration membrane with a pore size of 500 Da.

[0050] (10) The ultrafiltered camellia polysaccharide solution is concentrated by rotary evaporation, and the concentrate is dried or freeze-dried (to remove moisture) to obtain camellia polysaccharide.

[0051] (11) After testing, the yield of camellia polysaccharide was 1.09%, the purity was 65.78%, and the uronic acid content was 16.04%.

[0052] Example 2

[0053] (1) Crush and sieve camellia, and perform supercritical CO2 extraction at an extraction pressure of 30 MPa, an extraction temperature of 60° C., and an extraction time of 120 min. After the extraction is completed, take out the camellia powder to obtain defatted camellia powder;

[0054] (2) subjecting the defatted camellia powder to ultrasonic ethanol extraction to remove non-polysaccharide impurities such as flavonoids, polyphenols, and saponins. The ultrasonic extraction conditions are as follows: adding ethanol aqueous solution 30 times the mass of the defatted camellia powder, wherein the ethanol content is 100%, the ultrasonic temperature is controlled at 60° C., and then ultrasonically subjected to 180 minutes of ultrasonic power at 3000 W.

[0055] (3) After the ultrasonic treatment is completed, the temperature of the extract is cooled to room temperature, filtered to obtain the camellia powder after impurities are removed, and then dried;

[0056] (4) adding 5% by weight of cellulase and 30 times of water to the defatted and impurity-removed camellia powder in step (3) for ultrasonic extraction, controlling the aqueous solution to be below 40° C., and then ultrasonicating for 60 minutes, wherein the ultrasonic power is 3000 W;

[0057] (5) controlling the temperature of the above solution at 70° C., stirring and extracting for 3 h; then heating the solution to 95° C., stirring and extracting for 3 h, cooling the solution to room temperature, and filtering to obtain a camellia polysaccharide extract;

[0058] (6) Concentrating the camellia polysaccharide extract to 30% of the original mass, filtering through a 0.45 μm filter membrane to obtain a camellia polysaccharide concentrate;

[0059] (7) adding anhydrous ethanol to the above-mentioned camellia polysaccharide concentrate to precipitate the polysaccharide, so that the volume of ethanol accounts for 80% of the total system volume, at a temperature of 4° C. and a precipitation time of 12 h;

[0060] (8) filtering and drying the precipitate from step (7) to obtain crude camellia polysaccharide;

[0061] (9) Add water to the above-mentioned crude camellia polysaccharide to redissolve it so that the concentration of the crude camellia polysaccharide is 100 mg / ml, and perform ultrafiltration using an ultrafiltration membrane with a pore size of 10,000 Da.

[0062] (10) The ultrafiltered camellia polysaccharide solution is concentrated by rotary evaporation, and the concentrated solution is dried or freeze-dried to obtain camellia polysaccharide.

[0063] (11) After testing, the yield of camellia polysaccharide was 1.45%, the purity was 75.78%, and the uronic acid content was 26.04%.

[0064] Example 3

[0065] (1) Crush and sieve camellia, and perform supercritical CO2 extraction at an extraction pressure of 25 MPa, an extraction temperature of 50° C., and an extraction time of 120 min. After the extraction is completed, take out the camellia powder to obtain defatted camellia powder;

[0066] (2) subjecting the defatted camellia powder to ultrasonic ethanol extraction to remove non-polysaccharide impurities such as flavonoids, polyphenols, and saponins, wherein the ultrasonic extraction conditions are as follows: adding ethanol aqueous solution 20 times the mass of the defatted camellia powder, wherein the mass content of ethanol is 80%, the ultrasonic temperature is controlled at 50° C., and then ultrasonically subjected to 120 minutes of ultrasonication, wherein the ultrasonic power is 3000 W;

[0067] (3) After the ultrasonic treatment is completed, the temperature of the extract is cooled to room temperature, filtered to obtain the camellia powder after impurities are removed, and then dried;

[0068] (4) adding 1% by weight of cellulase and 20 times of water to the defatted and impurity-removed camellia powder in step (3) for ultrasonic extraction, controlling the aqueous solution to be below 40° C., and then ultrasonicating for 30 minutes, wherein the ultrasonic power is 3000 W;

[0069] (5) controlling the temperature of the solution at 60° C., stirring and extracting for 2 h, then heating the solution to 90° C., stirring and extracting for 2 h, cooling the solution to room temperature, and filtering to obtain a camellia polysaccharide extract;

[0070] (6) Concentrating the camellia polysaccharide extract to 20% of the original mass, filtering through a 0.45 μm filter membrane to obtain a camellia polysaccharide concentrate;

[0071] (7) adding anhydrous ethanol to the above-mentioned camellia polysaccharide concentrate to precipitate the polysaccharide, so that the volume of ethanol accounts for 60% of the total system volume, at a temperature of 4° C. and a precipitation time of 12 h;

[0072] (8) filtering and drying the precipitate from step (7) to obtain crude camellia polysaccharide;

[0073] (9) Add water to the above-mentioned crude camellia polysaccharide to redissolve it so that the concentration of the crude camellia polysaccharide is 50 mg / ml, and perform ultrafiltration using an ultrafiltration membrane with a pore size of 3500 Da.

[0074] (10) The ultrafiltered camellia polysaccharide solution is concentrated by rotary evaporation, and the concentrated solution is dried or freeze-dried to obtain camellia polysaccharide.

[0075] (11) After testing, the yield of camellia polysaccharide was 1.91%, the purity was 89.78%, and the uronic acid content was 35.04%.

[0076] Example 4

[0077] (1) Crush and sieve camellia, and perform supercritical CO2 extraction at an extraction pressure of 25 MPa, an extraction temperature of 50° C., and an extraction time of 120 min. After the extraction is completed, take out the camellia powder to obtain defatted camellia powder;

[0078] (2) subjecting the defatted camellia powder to ultrasonic ethanol extraction to remove non-polysaccharide impurities such as flavonoids, polyphenols, and saponins, wherein the ultrasonic extraction conditions are as follows: adding ethanol aqueous solution 20 times the mass of the defatted camellia powder, wherein the mass content of ethanol is 80%, the ultrasonic temperature is controlled at 50° C., and then ultrasonically subjected to 120 minutes of ultrasonication, wherein the ultrasonic power is 3000 W;

[0079] (3) After the ultrasonic treatment is completed, the temperature of the extract is cooled to room temperature, filtered to obtain the camellia powder after impurities are removed, and then dried;

[0080] (4) adding 1% by weight of cellulase and 20 times of water to the defatted and impurity-removed camellia powder in step (3) for ultrasonic extraction, controlling the aqueous solution to be below 40° C., and then ultrasonicating for 30 minutes, wherein the ultrasonic power is 3000 W;

[0081] (5) controlling the temperature of the above solution at 60° C. and stirring and extracting for 2 h; then heating the solution to 90° C. and stirring and extracting for 2 h, cooling the solution to room temperature, and filtering to obtain a camellia polysaccharide extract;

[0082] (6) Concentrating the camellia polysaccharide extract to 20% of the original mass, filtering through a 0.45 μm filter membrane to obtain a camellia polysaccharide concentrate;

[0083] (7) adding anhydrous ethanol to the above-mentioned camellia polysaccharide concentrate to precipitate the polysaccharide, so that the volume of ethanol accounts for 60% of the total system volume, at a temperature of 4° C. and a precipitation time of 12 h;

[0084] (8) filtering and drying the precipitate from step (7) to obtain crude camellia polysaccharide;

[0085] (9) Add water to the above-mentioned crude camellia polysaccharide to re-dissolve it so that the concentration of the crude camellia polysaccharide is 50 mg / ml, and perform ultrafiltration using an ultrafiltration membrane with a pore size of 5000 Da.

[0086] (10) The ultrafiltered camellia polysaccharide solution is concentrated by rotary evaporation, and the concentrated solution is dried or freeze-dried to obtain camellia polysaccharide.

[0087] (11) After testing, the yield of camellia polysaccharide was 1.83%, the purity was 88.11%, and the uronic acid content was 33.64%.

[0088] Comparative Example 1

[0089] This comparative example is a comparative example of Example 3, except that the supercritical CO2 defatting in step (1) of Example 3 is omitted, and other conditions remain unchanged. After testing, the yield of camellia polysaccharide is 0.65%, the purity is 39.25%, and the uronic acid content is 9.56%.

[0090] Comparative Example 2

[0091] This comparative example is a comparative example of Example 3. This comparative example omits the ultrasonic pretreatment in step (2) of Example 3 to remove non-sugar small molecule impurities such as saponins, flavonoids, and polyphenols, and other conditions remain unchanged. It was found through experiments that this step did not remove small molecule impurities such as saponins, and the subsequent concentration preparation was difficult, and the concentration could not be completed. A large amount of ethanol was required during alcohol precipitation. At the same time, very little polysaccharide was precipitated and it was difficult to recover. Considering industrial mass production, it did not have production conditions and production value.

[0092] Comparative Example 3

[0093] This comparative example is a comparative example of Example 3. In this comparative example, the ultrasonic pretreatment in steps (1) and (2) of Example 3 is omitted, and other conditions remain unchanged. This step does not perform the two steps of defatting and impurity removal, which not only affects the polysaccharide extraction effect but also makes the subsequent concentration and preparation of polysaccharides more difficult, and also does not meet the production conditions.

[0094] Comparative Example 4

[0095] This comparative example is a comparative example of Example 3, in which no cellulase is added in step (4) of this comparative example, and other conditions remain unchanged. After testing, the yield of camellia polysaccharide is 0.88%, the purity is 60.96%, and the uronic acid content is 18.56%.

[0096] Comparative Example 5

[0097] This comparative example is a comparative example of Example 3. In this comparative example, the extraction in step (5) of Example 3 is changed to a one-step extraction: heating to 90°C and stirring for extraction for 4 hours, with other conditions unchanged. After testing, the yield of camellia polysaccharide is 0.68%, the purity is 59.96%, and the uronic acid content is 10.56%.

[0098] Comparative Example 6

[0099] This comparative example is a comparative example of Example 3. In this comparative example, the extraction in step (5) of Example 3 is changed to a one-step extraction: heating to 60°C and stirring for extraction for 4 hours, with other conditions unchanged. After testing, the yield of camellia polysaccharide is 0.56%, the purity is 61.56%, and the uronic acid content is 16.56%.

[0100] Comparative Example 7

[0101] This comparative example is a comparative example of Example 3. In this comparative example, step (7) in Example 3 is omitted, and the extract in step (6) is directly concentrated, dried, and baked, and other conditions remain unchanged. After testing, the yield of camellia polysaccharide is 1.08%, the purity is 58.96%, and the uronic acid content is 15.56%.

[0102] Comparative Example 8

[0103] This comparative example is a comparative example of Example 3, in which steps (9) and (10) in Example 3 are omitted and other conditions remain unchanged. This comparative example can only obtain crude camellia polysaccharide with a yield of 3.40%, a purity of 30.88%, and a uronic acid content of 10.56%.

[0104] Efficacy verification experiment:

[0105] (1) Antioxidant test

[0106] The camellia polysaccharides prepared in all the above examples were subjected to antioxidant tests of DPPH free radical scavenging rate, and in order to more intuitively observe the antioxidant capacity of camellia polysaccharides, a common antioxidant Vc was selected as a control. The specific method is as follows:

[0107] Experimental steps: Add 2mL DPPH ethanol solution and 2mL test sample (Camellia polysaccharide 1-10mg / ml) solution into a test tube, shake and mix well, and react in the dark for 30 minutes. Measure the absorbance A at 517nm. At the same time, measure the absorbance A0 of the mixed solution of 2mL DPPH ethanol solution and 2mL anhydrous ethanol, and the absorbance Ab of the mixed solution of 2mL anhydrous ethanol and 2mL test sample.

[0108] Calculate the DPPH free radical scavenging rate: DPPH free radical scavenging rate (%) = (A0-(A-Ab)) / A0×100; IC50 is the half inhibition rate concentration: that is, the concentration of the free radical scavenger when the free radical scavenging rate is 50%. The IC50 value is a common indicator for evaluating the effect of free radical scavengers. The smaller the value, the smaller the concentration and dosage of the free radical scavenger used to achieve 50% free radical scavenging rate, and the better the free radical scavenging effect. After making the DPPH free radical scavenging action curve of the extract, the IC50 value is calculated to represent its antioxidant activity. The results are shown in Table 1 below:

[0109] Table 1 DPPH free radical IC50 values ​​of Camellia polysaccharides of Examples 1-4 and Comparative Examples 1-8

[0110] sample IC50(mg / mL) Example 1 8.25 Example 2 6.75 Example 3 6.25 Example 4 6.79 Comparative Example 1 18.47 Comparative Example 4 17.86 Comparative Example 5 18.35 Comparative Example 6 17.86 Comparative Example 7 16.35 Comparative Example 8 16.35

[0111] (2) Anti-wrinkle test

[0112] The camellia polysaccharides prepared in all the above examples were used to promote the synthesis of fibroblast type I collagen to verify the anti-wrinkle effect of the extract. The experimental method was based on T / SHRH 031-2020, Cosmetic firming and anti-wrinkle efficacy test-in vitro fibroblast type I collagen content determination, Shanghai Daily Cosmetics Industry Association, 2018.

[0113] Table 2 Promotion rate of camellia polysaccharide on type I collagen in Examples 1-4 and Comparative Examples 1-8

[0114] sample Addition concentration (mg / mL) Type Ⅰ collagen promotion rate (%) Example 1 1.0 26.26 Example 2 1.0 28.26 Example 3 1.0 29.56 Example 4 1.0 28.79 Comparative Example 1 1.0 12.25 Comparative Example 4 1.0 13.36 Comparative Example 5 1.0 15.34 Comparative Example 6 1.0 14.25 Comparative Example 7 1.0 18.36 Comparative Example 8 1.0 17.34

[0115] (3) Repair effect

[0116] Epidermal keratinocytes are important cells that constitute the epidermis. When the skin surface is damaged, keratinocytes will be stimulated to migrate and repair the damaged part. When the cells grow to a monolayer in vitro, a blank area is artificially created on the monolayer cells. The cells at the edge of the scratch will gradually enter the blank area to heal the scratch, simulating the cell migration process in vivo to a certain extent. By measuring the cell migration rate after sample treatment, it is evaluated whether the test substance has a repair effect. The efficacy of the composition was verified by testing the migration rate of epidermal keratinocytes prepared by all the examples.

[0117] Table 3 Effects of Camellia polysaccharides of Examples 1-4 and Comparative Examples 1-8 on cell migration rate

[0118] sample Addition concentration (mg / mL) Cell migration rate (%) Example 1 1.0 36.96 Example 2 1.0 38.66 Example 3 1.0 39.25 Example 4 1.0 38.79 Comparative Example 1 1.0 18.25 Comparative Example 4 1.0 21.44 Comparative Example 5 1.0 21.14 Comparative Example 6 1.0 22.25 Comparative Example 7 1.0 22.44 Comparative Example 8 1.0 19.14

[0119] Application Example 1

[0120] This application example provides the application and preparation method of camellia polysaccharide in the formulation of cosmetic water. The specific formula is as follows:

[0121] Table 4

[0122] Serial number Standard Chinese name content(%) 1 water TO 100 2 Butanediol 8.0 3 Xanthan gum 0.10 4 p-Hydroxyacetophenone 0.4 5 Disodium EDTA 0.05 6 1,2-Hexanediol 0.4 7 Camellia polysaccharide 3.5

[0123] The preparation method is as follows:

[0124] Add items 1, 2, 3, 4, and 5 to the main pot in sequence at room temperature, stir and disperse evenly, then heat to 85±2℃, homogenize for 3 minutes, and stir evenly;

[0125] Cool down to 60±2℃, add items 6 and 7 in sequence and stir evenly.

[0126] Application Example 2

[0127] This application example provides the application and preparation method of camellia polysaccharide in the essence formula. The specific formula is as follows:

[0128] Table 5

[0129]

[0130] The preparation method is as follows:

[0131] Add items 1, 2, 3, 4, 5, 6, and 7 to a water pot at room temperature, stir and disperse them evenly, then heat to 85±2℃, stir evenly, then pump into an emulsifying pot, homogenize for 3 minutes, and stir evenly;

[0132] Add items 8, 9 and 10 to the mixing bowl in sequence and stir well;

[0133] Add items 11, 12 and 13 into the oil pan in sequence at room temperature, then heat to 85±2℃, stir evenly and pump into the emulsification pot.

[0134] Homogenize for 5 minutes, keep warm for 10 minutes to eliminate foam, then add item 14, stir evenly, and keep for 10 minutes;

[0135] Cool down to 50±2℃, add items 15, 16 and 17 in sequence and stir evenly.

[0136] Application Example 3

[0137] This application example provides the application and preparation method of camellia polysaccharide in the formulation of skin care lotion. The specific formula is as follows:

[0138] Table 6

[0139] Serial number Standard Chinese name content(%) 1 water TO 100 2 glycerin 6.0 3 Butanediol 4.0 4 Sodium Hyaluronate 0.05 5 Xanthan gum 0.1 6 Ammonium Acryloyldimethyltaurate / VP Copolymer 0.35 7 p-Hydroxyacetophenone 0.5 8 Caprylic / capric triglyceride 3.0 9 Cetearyl Alcohol 0.8 10 Squalane 3.0 11 Murumuru palm seed butter, tocopherol (vitamin E) 1.5 12 Tocopheryl acetate 0.5 13 C14-22 Alcohol; C12-20 Alkyl Glucoside 1.5 14 Dimethicone 2.0 15 1,2-Hexanediol 0.4 16 Camellia polysaccharide 2 17 essence 0.1

[0140] The preparation method is as follows:

[0141] A. Add items 1, 2, 3, 4, 5, 6 and 7 into a water pot in sequence at room temperature, stir and disperse them evenly, then heat to 85±2℃, stir evenly and then pump into an emulsifying pot, homogenize for 3 minutes and stir evenly;

[0142] B. Add items 8, 9, 10, 11, 12, 13 and 14 into the oil pan in sequence at room temperature, then heat to 85±2℃. After they are completely dissolved, pump them into the emulsifying pot.

[0143] C. Homogenize for 8 minutes, keep warm for 10 minutes, and stir evenly;

[0144] D. Cool down to 50±2℃, add items 15, 16 and 17 and stir evenly.

[0145] Application Example 4

[0146] This application example provides the application and preparation method of camellia polysaccharide in the formulation of skin care cream. The specific formula is as follows:

[0147] Table 7

[0148]

[0149] The preparation method is as follows:

[0150] A. Add items 1, 2, 3, 4, 5 and 6 into a water pot in sequence at room temperature, stir and disperse them evenly, then heat to 85±2℃, stir evenly and then pump into an emulsifying pot, homogenize for 5 minutes and stir evenly;

[0151] B. Add items 7, 8, 9, 10, 11, 12, 13 and 14 into the oil pan in sequence at room temperature, then heat to 85±2℃. After they are completely dissolved, pump them into the emulsifying pot.

[0152] C. Homogenize for 8 minutes, keep warm for 10 minutes, and stir evenly;

[0153] D. Cool down to 50±2℃, add items 15, 16 and 17 in sequence and stir evenly.

[0154] Application Example 5

[0155] This application example provides the application and preparation method of camellia polysaccharide in the facial mask formulation. The specific formulation is as follows:

[0156] Table 8

[0157] Serial number Standard Chinese name content(%) 1 water TO 100 2 Butanediol 4.0 3 Glycereth-26 4.0 4 PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol 1.0 5 Sodium Polyglutamate 0.05 6 Sodium Hyaluronate 0.05 7 Sodium polyacrylate grafted starch 0.1 8 Dipotassium glycyrrhizinate 0.1 9 p-Hydroxyacetophenone 0.4 10 1,2-Hexanediol 0.3 11 Camellia polysaccharide 1.0

[0158] The preparation method is as follows:

[0159] A. Add items 1, 2, 3, 4, 5, 6, 7, 8 and 9 into a water pot in sequence at room temperature, stir and disperse them evenly, then heat to 85±2℃, stir evenly and then pump into an emulsifying pot, homogenize for 3 minutes and stir evenly;

[0160] B. Keep warm at 85±2℃ for 10 minutes and stir evenly;

[0161] C. Cool down to 50±2℃, add items 10 and 11 in sequence and stir evenly.

[0162] Application Example 6

[0163] This application example provides the application and preparation method of camellia polysaccharide in a facial cleanser formula. The specific formula is as follows:

[0164] Table 9

[0165]

[0166]

[0167] The preparation method is as follows:

[0168] A. Add items 1, 2, 3, 4, 5 and 6 into the emulsifying pot in sequence at room temperature, stir and disperse them evenly, then heat to 85±2℃ and stir evenly;

[0169] B. Add items 7 and 8 in sequence, keep warm for 20 minutes, and stir evenly;

[0170] C. Cool down to 50±2℃, add items 9, 10, 11 and 12 in sequence and stir evenly.

[0171] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A method for preparing camellia polysaccharide with anti-aging and repairing effects, characterized in that: The following steps are involved: A1. Crush and sieve camellia flowers, and perform supercritical CO2 extraction. After the extraction, take out the camellia powder to obtain defatted camellia powder; A2, subjecting the defatted camellia powder to ultrasonic extraction, cooling to room temperature, filtering, and drying to obtain the camellia powder after impurities are removed; A3, adding cellulase and water to the camellia powder after impurities are removed for ultrasonic extraction, then heating the extraction, cooling and filtering to obtain a camellia polysaccharide extract; A4, concentrating the camellia polysaccharide extract, filtering, and obtaining a camellia polysaccharide concentrate; A5, adding a solvent to the camellia polysaccharide concentrate to precipitate the polysaccharide, filtering, and drying the filter residue to obtain crude camellia polysaccharide; A6. Add water to the crude camellia polysaccharide to redissolve it, perform ultrafiltration, and concentrate the filtrate by rotary evaporation to obtain camellia polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step A1, the extraction pressure of supercritical CO2 extraction is 10 MPa-30 MPa, the extraction temperature is 35°C-60°C, and the extraction time is 30min-120min.

3. The preparation method according to claim 1, characterized in that: In step A2, the temperature of ultrasonic extraction is 40-60°C, the time is 30min-180min, and the ultrasonic power is 500W-3000W.

4. The preparation method according to claim 1, characterized in that: In step A3, the amount of cellulase used is 0.1-5% of the mass of the camellia powder after impurities are removed; And / or, the amount of water used is 10-30 times the mass of the camellia powder after impurities are removed.

5. The preparation method according to claim 1, characterized in that: In step A3, the ultrasonic power of ultrasonic extraction is 500W-3000W, and the ultrasonic time is 10min-60min; And / or, the temperature of the solution is controlled below 40° C. during ultrasonic extraction.

6. The preparation method according to claim 1, characterized in that: In step A3, the temperature-raising extraction is performed twice, the first temperature is raised to 40-70° C., and the extraction is performed for 1 h-3 h; the second temperature is raised to 75-95° C., and the extraction is performed for 0.5 h-3 h.

7. The preparation method according to claim 1, characterized in that: When precipitating polysaccharides, the temperature of the camellia polysaccharide concentrate is 1-4°C, and the precipitation time is 5-12h.

8. The preparation method according to claim 1, characterized in that: In the reconstituted solution, the concentration of camellia crude polysaccharide is 0.5-100 mg / ml; And / or, the ultrafiltration uses an ultrafiltration membrane, and the pore size of the ultrafiltration membrane is 500-10000Da.

9. Use of the camellia polysaccharide obtained by the preparation method as claimed in claim 1 in the preparation of cosmetics.

10. The use according to claim 9, characterized in that: The cosmetics include one of lotion, essence, skin care milk, skin care ointment, skin care cream, facial mask and facial cleansing preparation.

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