Preparation method of caulerpa lentillifera extract as well as product and application of caulerpa lentillifera extract

Abstract

The invention relates to the technical field of plant extraction, in particular to a preparation method of caulerpa lentillifera extract as well as a product and application of the caulerpa lentillifera extract, the preparation method of the caulerpa lentillifera extract at least comprises the following steps: S1, cleaning caulerpa lentillifera, draining, quick-freezing with liquid nitrogen, and performing superfine grinding to obtain caulerpa lentillifera powder; s2, mixing the caulerpa lentillifera powder with a composite anti-browning solution, performing gradient temperature-pressure vacuum extraction to obtain an extracting solution, and filtering the extracting solution to obtain a caulerpa lentillifera crude extracting solution; s3, performing pigment removal and centrifugation on the caulerpa lentillifera crude extract, taking supernate, performing membrane filtration and desalting treatment to obtain feed liquid, and performing membrane filtration and sterilization on the feed liquid to obtain the caulerpa lentillifera extract. The preparation method provided by the invention can effectively prevent tissue browning, the prepared caulerpa lentillifera extract has the functions of moisturizing and maintaining skin barrier, and the application of the caulerpa lentillifera in the field of cosmetics is expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and in particular to a preparation method of a long-stem grape fern algae extract, a product thereof and an application thereof. Background Art

[0002] Caulerpa lentillifera belongs to the genus Caulerpa, family Caulerpaceae, order Bryopsidales, order Chlorophyta. The chemical composition and efficacy of Caulerpa lentillifera are garnering increasing attention. For example, its polyphenols and alkaloids (phycoerythrin) possess strong antioxidant capacity, while the Caulerpa lentillifera polysaccharide, CLGP4, exhibits immunostimulatory activity and can exert anti-inflammatory activity by regulating the production of inflammatory factors. Both aqueous and alcoholic extracts of Caulerpa lentillifera exhibit significant inhibitory effects against Staphylococcus aureus and Streptococcus mutans. Consequently, Caulerpa lentillifera has become a research hotspot in the fields of health foods, pharmaceuticals, and cosmetics, demonstrating significant development potential.

[0003] Existing extracts of long-stemmed grape fern algae (sea grape extracts), such as Chinese invention patent application CN118078866A, disclose a method for preparing a sea grape extract, which includes: boiling sea grapes in 100°C water for extraction, collecting the supernatant, and freeze-drying the supernatant to obtain the sea grape extract. However, the extraction process can easily cause browning. Chinese invention patent application CN116785327A discloses a method for preparing a sea grape extract. However, the use of this sea grape extract in cosmetic formulations may cause skin allergies in consumers.

[0004] Furthermore, Chinese invention patent application CN114316081A prepares a sulfated polysaccharide from long-stemmed cauline cauline that inhibits SARS-CoV-2 by pulping it, hydrolyzing it with a neutral protease, extracting it with hot water, precipitating it with alcohol, removing proteins, and purifying it with chromatography and ultrafiltration. This preparation method easily releases polyphenol oxidase (PPO) and phenolic compounds into the water, making enzymatic browning very likely to occur during low-temperature hydrolysis. Summary of the Invention

[0005] In order to solve the problems in the prior art, the first aspect of the present invention provides a method for preparing a long-stem grape fern extract, comprising at least the following steps:

[0006] S1. Washing and filtering the long-stem grape cauliflower, and then quick-freezing the cauliflower with liquid nitrogen and ultrafine grinding to obtain long-stem grape cauliflower powder;

[0007] S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract to obtain a long-stem grape cauliflower crude extract;

[0008] S3. Depigmenting and centrifuging the crude extract of the long-stem grape cauliflower, taking the supernatant, filtering it through a membrane, and desalting it to obtain a feed liquid, and sterilizing the feed liquid through membrane filtration to obtain the long-stem grape cauliflower extract.

[0009] In one embodiment, the liquid nitrogen quick freezing conditions are: temperature -196°C to -100°C, time 15 to 180 seconds. The temperature can be -196°C, -150°C, -120°C, or -100°C; and the time can be 15 seconds, 30 seconds, 50 seconds, 100 seconds, 150 seconds, or 180 seconds.

[0010] In one embodiment, the particle size of the long stem grape fern algae powder is 200-500 mesh, which can be 200 mesh, 300 mesh, 400 mesh, or 500 mesh.

[0011] Polyphenol oxidase (PPO) is a copper-binding metalloproteinase widely found in cells such as plants, animals, and fungi. PPO is synthesized in the cytoplasm and is mainly located on the plastid membrane (chloroplasts, amyloplasts, etc.), while its substrate, phenolic substances, is mainly present in the vacuole, and the two show regional distribution. When the cell structure is destroyed (such as after crushing), the regional balance of enzyme and phenol is disrupted. In the presence of oxygen, PPO catalyzes the conversion of phenolic substances into quinones, which react with amines and sulfhydryl groups or self-polymerize to form melanin, thereby causing tissue browning. The present invention can effectively destroy the cell wall structure of long-stemmed grape fern algae through the combined action of liquid nitrogen quick freezing and ultrafine grinding, helping to reduce the damage to the components and preventing premature activation of polyphenol oxidase during the crushing process, effectively preventing tissue browning.

[0012] In one embodiment, the composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of ascorbic acid to citric acid is (1-5):1, which can be 1:1, 2:1, 3:1, 4:1, or 5:1.

[0013] The present invention limits the mass ratio of ascorbic acid to citric acid to (1-5):1, synergistically inhibiting both enzymatic and non-enzymatic browning through the dual mechanisms of acidification to inhibit enzyme activity and reduction to block oxidation. A high ascorbic acid ratio provides sufficient reducing power, while the low oxygen conditions of the gradient temperature-pressure vacuum extraction environment further enhance the ascorbic acid's reducing effect, effectively preventing the occurrence of browning. Furthermore, as natural ingredients, ascorbic acid and citric acid are used in the development of cosmetic raw materials, helping to ensure the color and quality of the extract.

[0014] In one embodiment, the amount of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae.

[0015] In one embodiment, the total mass concentration of ascorbic acid and citric acid in the composite anti-browning liquid is 1-4%, which can be 1%, 2%, 3%, or 4%.

[0016] In one embodiment, the gradient temperature and pressure vacuum extraction is performed under the protection of an inert gas, which is any one of nitrogen, helium, and argon.

[0017] In one embodiment, the gradient temperature-pressure vacuum extraction comprises three gradients:

[0018] First gradient, vacuum degree is 0.04-0.06MPa, temperature is 25-30℃, and time is 10-20min;

[0019] The second gradient is vacuum degree of 0.06-0.085 MPa, temperature of 28-32°C, and time of 20-30 min;

[0020] The third gradient is a vacuum degree of 0.085-0.099 MPa, a temperature of 30-35°C, and a time of 30-60 min.

[0021] The present invention adopts a gradient temperature and pressure extraction process. By gradually reducing the system pressure, the osmotic pressure balance in the cells is broken, thereby gradually releasing the water in different bound states in the long-stem grape fern algae, accelerating the penetration and dissolution of intracellular substances such as polysaccharides, thereby shortening the extraction time and improving the extraction efficiency. Its advantages are reflected in the following aspects: 1) By step-by-step pressure reduction and temperature increase, the material boiling phenomenon can be avoided, and the extraction effect equivalent to or even better than that of normal pressure and high temperature can be achieved under low temperature conditions (<35°C), while maintaining the integrity of active ingredients such as β-glycosidic bonds in polysaccharides, avoiding high temperature damage to the structures of polysaccharides, proteins, polyphenols, etc., and maximizing the retention of heat-sensitive active ingredients. 2) Under the dual effects of the inert gas environment and the formation of a reduced state protection system by ascorbic acid and polyphenols, the risk of active ingredients being oxidized during the extraction process is significantly reduced, further protecting the biological activity and stability of the long-stem grape fern algae components. 3) The extraction process uses the long-stem grape fern algae cells' own water, that is, liquid cell water, to dissolve the active ingredients in the raw materials, reducing the introduction of exogenous water, which is in line with the concept of pure beauty.

[0022] In one embodiment, filtration is performed using a 0.1 μm ceramic membrane in step S2.

[0023] In one embodiment, the centrifugation conditions in step S3 are: 4° C., 5000 rpm, 15 min.

[0024] In one embodiment, in step S3, the pigment of the crude extract of long-stem grape ferns is removed by activated carbon and a clarifier, the amount of activated carbon used is 0.05-1% of the mass of the crude extract of long-stem grape ferns, and the amount of the clarifier used is 1-5% of the mass of the crude extract of long-stem grape ferns.

[0025] In one embodiment, the clarifier includes one or more of chitosan, ZTC-II type clarifier, ZTC-III type clarifier, and 101 type clarifier.

[0026] In one embodiment, in step S3, cation exchange resin and anion exchange resin chromatography are used for desalting.

[0027] In one embodiment, the type of the cation exchange resin is selected from one of 001×7, HD-2, D155, and D061.

[0028] In one embodiment, the model of the cation exchange resin is 001×7.

[0029] In one embodiment, the anion exchange resin is selected from one of 201×7, D941, D151, and D113.

[0030] In one embodiment, the anion exchange resin is of type D113.

[0031] In one embodiment, the dynamic adsorption flow rate of the cationic resin is 4 to 12 BV / h, and the dynamic adsorption flow rate of the anionic resin is 4 to 10 BV / h.

[0032] In one embodiment, the desalination treatment is specifically as follows: the supernatant is filtered through a membrane, and then filtered through a cationic resin for adsorption at a flow rate of 4 to 12 BV / h, and then filtered through an anion exchange resin for adsorption at a flow rate of 4 to 10 BV / h to obtain a feed solution.

[0033] A second aspect of the present invention provides a long-stem grape fern extract prepared according to the above preparation method.

[0034] A third aspect of the present invention provides a use of a long-stem grape cauliflower extract, wherein the long-stem grape cauliflower extract is used in the preparation of cosmetics.

[0035] Beneficial effects

[0036] 1. The present invention can effectively destroy the cell wall structure of the long-stem grape fern algae through the combined effects of liquid nitrogen quick freezing and ultrafine grinding, which helps to reduce the damage to the components and prevent the premature activation of polyphenol oxidase during the grinding process, effectively preventing tissue browning.

[0037] 2. The present invention limits the mass ratio of ascorbic acid to citric acid to (1-5):1, synergistically inhibiting both enzymatic and non-enzymatic browning through the dual mechanisms of acidification (inhibiting enzyme activity) and reduction (blocking oxidation). Furthermore, the low oxygen conditions of the gradient temperature-pressure vacuum extraction environment further enhance the reducing effect of ascorbic acid, effectively preventing the occurrence of browning.

[0038] 3. The present invention adopts a gradient temperature-pressure extraction process. Through step-by-step pressure reduction and temperature increase, it avoids the damage of high temperature to the structures of polysaccharides, proteins, polyphenols, etc., maximizes the retention of heat-sensitive active ingredients, and further protects the biological activity and stability of the long-stem grape fern components. In addition, the extraction process utilizes the water content of the long-stem grape fern cells themselves, that is, liquid cell water, to dissolve the active ingredients in the raw materials, reducing the introduction of exogenous water, which is in line with the concept of pure beauty.

[0039] 4. The long-stemmed grape fern extract prepared by this invention is rich in polysaccharides, proteins, polyphenols, and other ingredients. It has moisturizing and skin barrier maintenance functions, including the following: ① significant DPPH free radical scavenging ability; ② promoting hyaluronic acid secretion in HaCaT cells; ③ increasing the expression of involucrin (IVL) mRNA, a key protein in cornified envelope formation, in NHEK cells; ④ promoting the production of glycosaminoglycans (GAGs) in HSF cells; and ⑤ clinically demonstrating barrier repair effects. This expands the application of long-stemmed grape fern in the cosmetics field.

[0040] 5. The preparation method provided by the present invention avoids the use of high-temperature treatment and does not involve the use of organic solvents, thus avoiding the browning phenomenon during the extraction process, and has good application prospects in the preparation of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Graphs showing the test results of hyaluronic acid secretion by HaCaT cells in various embodiments and comparative examples.

[0042] Figure 2 The figure is a comparison of the effects of various embodiments and comparative examples on the expression of IVL genes in NHEK cells.

[0043] Figure 3 The figure is a comparison of the effects of various examples and comparative examples on the GAGs content in HSF cells.

[0044] Figure 4 This is a comparison chart of the skin TEWL change test results of the samples of Comparative Example 3 and Example 5. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods in the examples where specific conditions are not specified were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents used where the manufacturer is not specified are all conventional products that can be purchased commercially.

[0046] Example 1

[0047] The first aspect of this example provides a method for preparing a long-stem grape fern extract, comprising the following steps:

[0048] S1. Take fresh long-stem grape ferns and rinse with running water to remove surface attachments and other impurities, filter and dry, then quickly freeze with liquid nitrogen and ultrafinely grind to obtain long-stem grape ferns powder;

[0049] S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a long-stem grape cauliflower crude extract;

[0050] S3. Activated carbon was added to the crude extract of the long-stem grape fern algae, and the mixture was stirred evenly. After standing for 30 minutes, a clarifier was added. After standing for 30 minutes, the mixture was centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm polypropylene membrane, and then adsorbed and filtered through a cationic resin at a flow rate of 4 BV / h. The supernatant was then adsorbed and filtered through an anion exchange resin at a flow rate of 6 BV / h to obtain a feed liquid. The feed liquid was filtered and sterilized through a 0.22 μm polypropylene membrane to obtain the long-stem grape fern algae extract.

[0051] The liquid nitrogen quick freezing condition is -196°C for 30s.

[0052] The particle size of the ultrafinely pulverized long-stem grape fern algae powder is 200 meshes.

[0053] The composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of the ascorbic acid to the citric acid is 3:1.

[0054] The dosage of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae. The total mass concentration of ascorbic acid and citric acid in the composite anti-browning liquid is 2%.

[0055] The gradient temperature and pressure vacuum extraction is specifically as follows: under nitrogen protection, extraction is carried out at a vacuum degree of 0.05 MPa and 28° C. for 15 minutes; then extraction is carried out at a vacuum degree of 0.075 MPa and 30° C. for 25 minutes; and finally extraction is carried out at a vacuum degree of 0.095 MPa and 32° C. for 45 minutes.

[0056] The amount of activated carbon used is 0.5% of the mass of the crude extract of long-stem grape cauliflower, and the amount of clarifier used is 2% of the mass of the crude extract of long-stem grape cauliflower. The clarifier is a ZTC-II type clarifier.

[0057] The model of the cation exchange resin is 001×7. The model of the anion exchange resin is D113.

[0058] In a second aspect, this example provides a long-stem grape fern extract prepared according to the above preparation method.

[0059] The third aspect of this example provides an application of a long-stem grape cauliflower extract, wherein the long-stem grape cauliflower extract is used in the preparation of cosmetics.

[0060] Example 2

[0061] The first aspect of this example provides a method for preparing a long-stem grape fern extract, comprising the following steps:

[0062] S1. Take fresh long-stem grape ferns and rinse with running water to remove surface attachments and other impurities, filter and dry, then quickly freeze with liquid nitrogen and ultrafinely grind to obtain long-stem grape ferns powder;

[0063] S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a long-stem grape cauliflower crude extract;

[0064] S3. Activated carbon was added to the crude extract of the long-stem grape fern algae, and the mixture was stirred evenly. After standing for 30 minutes, a clarifier was added. After standing for 30 minutes, the mixture was centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm polypropylene membrane, and then adsorbed and filtered through a cationic resin at a flow rate of 4 BV / h. The supernatant was then adsorbed and filtered through an anion exchange resin at a flow rate of 6 BV / h to obtain a feed liquid. The feed liquid was sterilized by membrane filtration to obtain the long-stem grape fern algae extract.

[0065] The liquid nitrogen quick freezing condition is -120°C for 120s.

[0066] The particle size of the ultrafinely pulverized long-stem grape fern algae powder is 200 meshes.

[0067] The composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of the ascorbic acid to the citric acid is 3:1.

[0068] The dosage of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae. The total mass concentration of ascorbic acid and citric acid in the composite anti-browning liquid is 2%.

[0069] The gradient temperature and pressure vacuum extraction is specifically as follows: under nitrogen protection, extraction is carried out at a vacuum degree of 0.05 MPa and 28° C. for 15 minutes; then extraction is carried out at a vacuum degree of 0.075 MPa and 30° C. for 25 minutes; and finally extraction is carried out at a vacuum degree of 0.095 MPa and 32° C. for 45 minutes.

[0070] The amount of activated carbon used is 0.5% of the mass of the crude extract of long-stem grape cauliflower, and the amount of clarifier used is 2% of the mass of the crude extract of long-stem grape cauliflower. The clarifier is a ZTC-II type clarifier.

[0071] The model of the cation exchange resin is 001×7. The model of the anion exchange resin is D113.

[0072] In a second aspect, this example provides a long-stem grape fern extract prepared according to the above preparation method.

[0073] The third aspect of this example provides an application of a long-stem grape cauliflower extract, wherein the long-stem grape cauliflower extract is used in the preparation of cosmetics.

[0074] Example 3

[0075] The first aspect of this example provides a method for preparing a long-stem grape fern extract, comprising the following steps:

[0076] S1. Take fresh long-stem grape ferns and rinse with running water to remove surface attachments and other impurities, filter and dry, then quickly freeze with liquid nitrogen and ultrafinely grind to obtain long-stem grape ferns powder;

[0077] S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a long-stem grape cauliflower crude extract;

[0078] S3. Activated carbon was added to the crude extract of the long-stem grape fern algae, and the mixture was stirred evenly. After standing for 30 minutes, a clarifier was added. After standing for 30 minutes, the mixture was centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm polypropylene membrane, and then adsorbed and filtered through a cationic resin at a flow rate of 4 BV / h. The supernatant was then adsorbed and filtered through an anion exchange resin at a flow rate of 6 BV / h to obtain a feed liquid. The feed liquid was sterilized by membrane filtration to obtain the long-stem grape fern algae extract.

[0079] The liquid nitrogen quick freezing condition is -196°C for 30s.

[0080] The particle size of the ultrafinely pulverized long-stem grape fern algae powder is 200 meshes.

[0081] The composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of the ascorbic acid to the citric acid is 1:1.

[0082] The dosage of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae. The total mass concentration of ascorbic acid and citric acid in the composite anti-browning liquid is 2%.

[0083] The gradient temperature and pressure vacuum extraction is specifically as follows: under nitrogen protection, extraction is carried out at a vacuum degree of 0.05 MPa and 28° C. for 15 minutes; then extraction is carried out at a vacuum degree of 0.075 MPa and 30° C. for 25 minutes; and finally extraction is carried out at a vacuum degree of 0.095 MPa and 32° C. for 45 minutes.

[0084] The amount of activated carbon used is 0.5% of the mass of the crude extract of long-stem grape cauliflower, and the amount of clarifier used is 2% of the mass of the crude extract of long-stem grape cauliflower. The clarifier is a ZTC-II type clarifier.

[0085] The model of the cation exchange resin is 001×7. The model of the anion exchange resin is D113.

[0086] In a second aspect, this example provides a long-stem grape fern extract prepared according to the above preparation method.

[0087] The third aspect of this example provides an application of a long-stem grape cauliflower extract, wherein the long-stem grape cauliflower extract is used in the preparation of cosmetics.

[0088] Example 4

[0089] The first aspect of this example provides a method for preparing a long-stem grape fern extract, comprising the following steps:

[0090] S1. Take fresh long-stem grape ferns and rinse with running water to remove surface attachments and other impurities, filter and dry, then quickly freeze with liquid nitrogen and ultrafinely grind to obtain long-stem grape ferns powder;

[0091] S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a long-stem grape cauliflower crude extract;

[0092] S3. Activated carbon was added to the crude extract of the long-stem grape fern algae, and the mixture was stirred evenly. After standing for 30 minutes, a clarifier was added. After standing for 30 minutes, the mixture was centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm polypropylene membrane, and then adsorbed and filtered through a cationic resin at a flow rate of 4 BV / h. The supernatant was then adsorbed and filtered through an anion exchange resin at a flow rate of 6 BV / h to obtain a feed liquid. The feed liquid was sterilized by membrane filtration to obtain the long-stem grape fern algae extract.

[0093] The liquid nitrogen quick freezing condition is -196°C for 30s.

[0094] The particle size of the ultrafinely pulverized long-stem grape fern algae powder is 200 meshes.

[0095] The composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of the ascorbic acid to the citric acid is 3:1.

[0096] The dosage of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae. The total mass concentration of ascorbic acid and citric acid in the composite anti-browning liquid is 2%.

[0097] The gradient temperature and pressure vacuum extraction is specifically as follows: under nitrogen protection, extraction is carried out at a vacuum degree of 0.04 MPa and 28° C. for 15 minutes; then extraction is carried out at a vacuum degree of 0.06 MPa and 32° C. for 25 minutes; and finally extraction is carried out at a vacuum degree of 0.09 MPa and 35° C. for 45 minutes.

[0098] The amount of activated carbon used is 0.5% of the mass of the crude extract of long-stem grape cauliflower, and the amount of clarifier used is 2% of the mass of the crude extract of long-stem grape cauliflower. The clarifier is a ZTC-II type clarifier.

[0099] The model of the cation exchange resin is 001×7. The model of the anion exchange resin is D113.

[0100] In a second aspect, this example provides a long-stem grape fern extract prepared according to the above preparation method.

[0101] The third aspect of this example provides an application of a long-stem grape cauliflower extract, wherein the long-stem grape cauliflower extract is used in the preparation of cosmetics.

[0102] Example 5

[0103] The first aspect of this example provides a method for preparing a long-stem grape fern extract, comprising the following steps:

[0104] S1. Take fresh long-stem grape ferns and rinse with running water to remove surface attachments and other impurities, filter and dry, then quickly freeze with liquid nitrogen and ultrafinely grind to obtain long-stem grape ferns powder;

[0105] S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a long-stem grape cauliflower crude extract;

[0106] S3. Activated carbon was added to the crude extract of the long-stem grape fern algae, and the mixture was stirred evenly. After standing for 30 minutes, a clarifier was added. After standing for 30 minutes, the mixture was centrifuged at 4°C and 5000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm polypropylene membrane, and then adsorbed and filtered through a cationic resin at a flow rate of 4 BV / h. The supernatant was then adsorbed and filtered through an anion exchange resin at a flow rate of 6 BV / h to obtain a feed liquid. The feed liquid was sterilized by membrane filtration to obtain the long-stem grape fern algae extract.

[0107] The liquid nitrogen quick freezing condition is -196°C for 30s.

[0108] The particle size of the ultrafinely pulverized long-stem grape fern algae powder is 200 meshes.

[0109] The composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of the ascorbic acid to the citric acid is 3:1.

[0110] The dosage of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae. The total mass concentration of ascorbic acid and citric acid in the composite anti-browning liquid is 2%.

[0111] The gradient temperature and pressure vacuum extraction is specifically as follows: under nitrogen protection, extraction is carried out at a vacuum degree of 0.06 MPa and 25° C. for 15 minutes; then extraction is carried out at a vacuum degree of 0.085 MPa and 28° C. for 25 minutes; and finally extraction is carried out at a vacuum degree of 0.096 MPa and 30° C. for 45 minutes.

[0112] The amount of activated carbon used is 0.5% of the mass of the crude extract of long-stem grape cauliflower, and the amount of clarifier used is 2% of the mass of the crude extract of long-stem grape cauliflower. The clarifier is a ZTC-II type clarifier.

[0113] The model of the cation exchange resin is 001×7. The model of the anion exchange resin is D113.

[0114] In a second aspect, this example provides a long-stem grape fern extract prepared according to the above preparation method.

[0115] The third aspect of this example provides an application of a long-stem grape cauliflower extract, wherein the long-stem grape cauliflower extract is used in the preparation of cosmetics.

[0116] Comparative Example 1

[0117] The specific implementation method of this example is the same as that of Example 1, except that step S1 is as follows: fresh long-stem grape fern algae is rinsed with running water to remove surface attachments and other impurities, filtered and then slurried using a homogenizer at a speed of 8000 rpm for 30 seconds each time, divided into 3 operations, with an interval of 20 seconds between each operation.

[0118] The remaining steps are consistent with those in Example 1.

[0119] Comparative Example 2

[0120] The specific implementation of this example is the same as that of Example 1, except that no composite anti-browning liquid is added in step S2.

[0121] The remaining steps are consistent with those in Example 1.

[0122] Comparative Example 3

[0123] The specific implementation method of this example is the same as that of Example 1, except that step S2 is as follows: mixing the long-stem grape fern algae powder with the composite anti-browning liquid and performing vacuum extraction under nitrogen protection at a vacuum degree of 0.095 MPa and 32° C. for 85 minutes to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a crude extract of long-stem grape fern algae.

[0124] The remaining steps are consistent with those in Example 1.

[0125] Figure 4 This is a comparison chart of the skin TEWL change test results of the samples of Comparative Example 3 and Example 5.

[0126] Comparative Example 4

[0127] The specific implementation of this comparative example is the same as that of Example 3, except that step S1 comprises: taking fresh long-stem grape fern algae and rinsing it with running water to remove surface attachments and other impurities, filtering it to dryness, freeze-drying it, and ultrafine grinding it to obtain 200-mesh long-stem grape fern algae powder;

[0128] The step S2 comprises: putting the long-stem grape cauliflower powder into an extraction tank, extracting with boiling water at 100° C. for 85 minutes to obtain an extract, and filtering the extract through a 0.1 μm ceramic membrane to obtain a long-stem grape cauliflower crude extract.

[0129] The remaining steps are consistent with those in Example 1.

[0130] Figure 1 Graph showing the test results of hyaluronic acid secretion by HaCaT cells in various embodiments and comparative examples; Figure 2 Graph comparing the effects of various examples and comparative examples on NHEK cell IVL gene expression; Figure 3 The figure is a comparison of the effects of various examples and comparative examples on the GAGs content in HSF cells.

[0131] Performance Testing

[0132] 1. Browning index determination:

[0133] 2.0 mL of each of the examples and comparative examples' extracts were taken, and 2.0 mL of 95% ethanol (volume fraction) was added to each sample. The mixture was then shaken for 20 minutes and centrifuged at 12,000 rpm for 5 minutes. The supernatant was collected and its absorbance was measured at 420 nm using a visible spectrophotometer. Three replicates were performed. The absorbance was used to represent the browning index of the sample, with a higher value indicating more severe browning. The test results are shown in Table 1.

[0134] 2. Solid content determination:

[0135] The solid content of the long-stemmed cauline extracts obtained in each example and comparative example was measured using a moisture meter (Ohaus, MB120). The meter's lid was opened, an empty sample tray was placed on the tray handle, the lid was closed, and the meter was peeled. The lid was opened again, and approximately 1.0 g of the example or comparative example sample was added. The lid was closed, and the moisture meter began automatic drying and measurement. After the test, the solid content was recorded. The test results are shown in Table 1.

[0136] Table 1

[0137]

[0138]

[0139] Absorbance at 420 nm is generally a good indicator of the production of water-soluble brown pigments. Absorbance at this wavelength is considered an important indicator of the extent of both enzymatic and non-enzymatic browning. Comparing the browning indices of Example 1 with Comparative Examples 2 and 4 in Table 1 reveals that high-temperature extraction causes significant browning in the long-stemmed cauline extract. However, rapid freezing with liquid nitrogen, treatment with a composite anti-browning solution, and inert gas protection significantly reduce changes in the browning index during extraction.

[0140] In the solid content determination, Example 5 had the best effect, followed by Example 1. Although Comparative Example 4 had a higher solid content, its browning index was too high, which limited its stability and application potential in cosmetic products.

[0141] 3. DPPH free radical scavenging activity assay:

[0142] 1) Preparation of DPPH solution: Weigh an appropriate amount of DPPH powder and dissolve it in ethanol to prepare a 0.2 mM DPPH solution.

[0143] 2) Positive control solution preparation: Weigh an appropriate amount of L-ascorbic acid powder and dissolve in distilled water to prepare a 10 mg / mL L-ascorbic acid solution. Dilute with ethanol to a final experimental concentration of 5 μg / mL.

[0144] 3) Preparation of test sample diluent: dilute the test sample (the extract of the long-stemmed cauline algae prepared in each example and comparative example) with ethanol to a final experimental concentration of 3%.

[0145] 4) DPPH Radical Scavenging Test: Add 500 μL each of the diluted sample solution and the positive control solution to a 24-well plate (three replicates). Add 500 μL of 0.2 mM DPPH solution to one well to compensate for the sample's absorbance, and add 500 μL of ethanol to the other well. Incubate at room temperature, protected from light, for 30 minutes. Measure absorbance at 517 nm, and calculate DPPH scavenging efficiency.

[0146] Table 2

[0147]

[0148]

[0149] The polysaccharides, polyphenols, and phycoerythrins of long-stem grape fern have strong free radical scavenging activity. However, during the extraction process, the occurrence of browning will affect the stability of the above components and have an adverse effect on their free radical scavenging efficiency.

[0150] In the results of the DPPH free radical scavenging rate determination, the effect shown by Example 5 is comparable to that of the positive drug L-ascorbic acid, and has the best DPPH scavenging ability, followed by Example 1. In contrast, Comparative Examples 1, 2, and 4 have different degrees of browning, resulting in a significant reduction in their DPPH free radical elimination effect. Overall, the effect of the embodiment is better than that of the comparative example. 4. HaCaT cell hyaluronic acid secretion test:

[0151] HaCaT cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% double antibody in a constant temperature incubator at 37°C and 5% CO2. Cells in logarithmic growth phase were taken and cell suspension was prepared at 1×10 5 The cells were seeded at a density of 1000 cells / well in a 24-well plate and incubated in a CO2 constant temperature incubator. After culturing for 24 hours, culture medium containing 3% concentration of the example and comparative example samples was added to each well of the sample group except the control group, 100 ng / mL retinoic acid was added to each well of the positive drug group, and 500 μL cell culture medium was added to each well of the control group. After the administration was completed, the 24-well plate was placed at 37°C and 5% CO2 and cultured for 24 hours. The upper culture medium was collected, centrifuged at 12000 rpm for 3 minutes, and the hyaluronic acid content was detected according to the instructions of the ELISA kit (R&D Systems). The test results are shown in Figure 1 .

[0152] The results of the hyaluronic acid (HA) content test showed that the positive drug retinoic acid significantly increased the HA content of cells compared with the control group. At the tested concentrations, Examples 1 to 5 significantly promoted the secretion of HA by HaCaT cells, with Example 5 and Example 1 showing the best results. Overall, the results of the Examples were superior to those of the Control Group.

[0153] 5. Detection of IVL (Involucrin) expression level in NHEK cells:

[0154] NHEK cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% double antibody in a constant temperature incubator at 37°C and 5% CO2. Cells in logarithmic growth phase were taken and cell suspension was prepared at 7.5×10 4 The cells were seeded at a density of 100 cells / well in a 6-well plate and incubated in a CO2 constant temperature incubator. After culturing for 24 hours, culture medium containing 3% concentration of the example and comparative example samples was added to each well of the sample group except the control group, and 2.5 mL of cell culture medium was added to each well of the control group. After the administration was completed, the 6-well plate was placed at 37°C and 5% CO2 and cultured for 72 hours. The cells were washed twice with PBS, and RNA was extracted according to the kit instructions. After reverse transcription to cDNA, fluorescence quantitative PCR was performed, and 2 -ΔΔct The relative expression of IVL was analyzed by the method of Figure 2 .

[0155] IVL (involucrin) is an early precursor protein of the epidermal cornified envelope and serves as a scaffold for the attachment of other cornified envelope proteins. Therefore, IVL is a key protein involved in the formation of the cornified envelope and plays an irreplaceable role in maintaining the skin barrier function. Figure 2 It can be seen that the IVL mRNA expression level in NHEK cells treated with Examples 1 to 5 was significantly higher than that in the blank control group, and the overall effect was better than that of Comparative Examples 1 to 4.

[0156] 6. Detection of GAGs (glycosaminoglycans) content in HSF cells:

[0157] HSF cells were cultured in a high-glucose DMEM medium containing 15% fetal bovine serum and 1% streptomycin / penicillin at 37°C in a cell culture incubator containing 5% CO2. The medium was changed and passaged regularly, and cells in the logarithmic growth phase were used for experiments. 4Cells were seeded in a 24-well plate at a density of 1000 cells / well and cultured for 24 hours. Culture fluid containing 3% concentration of the example and comparative example samples was added to each well of the sample group, and 500 μL of cell culture fluid was added to each well of the control group. After the administration was completed, the 24-well plate was placed at 37°C and 5% CO2 and cultured for 48 hours. After the culture was completed, the GAGs content in the cell supernatant was detected by DMB (1,9-dimethylmethylene blue) staining. The cell supernatant was digested with 0.5 mg / ml papain at 65°C for 2 hours, and centrifuged at 10,000 r / m for 8 minutes after digestion. DMB solution was then added to the cell supernatant digestion solution, and the absorbance value was detected at a wavelength of 520 nm. Chondroitin sulfate standard solutions of different mass concentrations were taken and added to the DMB solution, and the absorbance was detected at a wavelength of 520 nm to draw a standard curve for GAGs content. The absorbance value of each experimental group was compared with the standard curve to calculate the GAGs content. See the test results. Figure 3 .

[0158] Glycosaminoglycans (GAGs) are the main components of the dermis, mostly present in the form of proteoglycans in cell tissues, and then connected to collagen in a specific way, thus giving the matrix a special structure to maintain its stability. Figure 3 It can be seen that the amount of GAGs produced in HSF cells after treatment with Examples 1 to 5 is significantly higher than that in the blank control group, with Examples 5 and 1 having the best effects, and the effects of Examples 1 to 5 are generally better than those of Comparative Examples 1 to 4.

[0159] 7. Skin patch safety test:

[0160] Referring to the skin occlusive patch test method in the "Technical Specifications for Safety of Cosmetics" (2015 edition), 30 subjects aged 20 to 55 years old were selected to meet the conditions. Example 1 and Example 5 were prepared with distilled water to prepare sample solutions with mass fractions of 1%, 2%, and 3%, respectively. 2 , a spot test device with a depth of about 1mm. 20μL of sample solution was added to the small chamber of the spot tester respectively, and the blank control group was distilled water of the same dose. The spot tester with the test substance was applied to the curved side of the subject's forearm with low-allergenic tape, and gently pressed with the palm of the hand to evenly apply it to the skin for 24 hours. After removing the test substance spot tester, observe the skin reaction according to the standards in Table 3 at 30 minutes (after the indentation disappears), 24 hours and 48 hours, and record the observation results. The test results are shown in Table 4. The results of the human skin patch test showed that there was no adverse skin reaction among the 30 people.

[0161] Table 3

[0162]

[0163] Table 4

[0164]

[0165]

[0166] 8. Clinical barrier repair effect test:

[0167] The subjects were women or men (N=17), aged 18 to 60, who reported experiencing symptoms of impaired skin barrier function, such as dryness, scaling, and redness. After washing their hands and face and patting their skin dry with a paper towel, they entered the testing environment (constant temperature and humidity: 22±2°C, 50±10% RH) and rested for 30 minutes before entering the test.

[0168] The sample was used by applying 3% of the base gel of Example 5 and Comparative Example 3 to each half of the face (once in the morning and once in the evening) for 4 weeks. The transepidermal water loss (TEWL) was measured using a VapoMeter on day 0 (D0), day 14 (D14), and day 28 (D28). The average value of the three replicate measurements was obtained. The test results are shown in Table 1. Figure 4 (Statistical differences of the test data were analyzed simultaneously, compared with D0 day, *P<0.05, **P<0.01, ***P<0.001).

[0169] The formula of the base gel is as follows, by weight percentage: 3% of the long-stem grape fern extract prepared in Example 5 or Comparative Example 3; 0.02% of EDTA-2Na; 0.5% of carbomer (Carbopol 940 Polymer); 0.8% of Raysoncare BHC (octanoylhydroxamic acid, 1,2-hexanediol, butanediol); an appropriate amount of NaOH; and water to 100%.

[0170] like Figure 4 As shown, when subjects applied a base gel containing 3% of the long-stem cauliflower extract (Example 5) to their faces, transepidermal water loss (TEWL) was significantly reduced at 14 and 28 days, with reductions of 11.71% and 17.39%, respectively, compared to day 0. This indicates that the long-stem cauliflower extract has a barrier-repairing effect. In contrast, after using the base gel of Comparative Example 3, the maximum reduction in TEWL compared to the initial value was 9.40%, which was significantly less effective than the Example.

Claims

1. A method for preparing a long-stem grape fern extract, characterized in that: At least the following steps are included: S1. Washing and filtering the long-stem grape cauliflower, and then quick-freezing the cauliflower with liquid nitrogen and ultrafine grinding to obtain long-stem grape cauliflower powder; S2, mixing the long-stem grape cauliflower powder with the composite anti-browning liquid, and performing gradient temperature-pressure vacuum extraction to obtain an extract, and filtering the extract to obtain a long-stem grape cauliflower crude extract; S3. Depigmenting and centrifuging the crude extract of the long-stem grape cauliflower, taking the supernatant, filtering it through a membrane, and desalting it to obtain a feed liquid, and sterilizing the feed liquid through membrane filtration to obtain the long-stem grape cauliflower extract.

2. The method for preparing the long-stem grape fern extract according to claim 1, characterized in that: The liquid nitrogen quick freezing conditions are: temperature -196° C. to -100° C., time 15 to 180 seconds.

3. The method for preparing the long-stem grape fern extract according to claim 2, characterized in that: The particle size of the long-stem grape fern algae powder is 200-500 meshes.

4. The method for preparing the long-stem grape fern extract according to claim 1, characterized in that: The composite anti-browning liquid is a combination of ascorbic acid and citric acid, and the mass ratio of the ascorbic acid to the citric acid is (1-5):

1.

5. The method for preparing the long-stem grape fern extract according to claim 4, characterized in that: The dosage of the composite anti-browning liquid is 5% of the mass of the long-stem grape fern algae.

6. The method for preparing the long-stem grape fern extract according to claim 1, characterized in that: The gradient temperature and pressure vacuum extraction includes three gradients: First gradient, vacuum degree is 0.04-0.06MPa, temperature is 25-30℃, and time is 10-20min; The second gradient is vacuum degree of 0.06-0.085 MPa, temperature of 28-32°C, and time of 20-30 min; The third gradient is a vacuum degree of 0.085-0.099 MPa, a temperature of 30-35°C, and a time of 30-60 min.

7. The method for preparing the long-stem grape fern extract according to claim 1, characterized in that: In step S3, the pigment of the crude extract of long-stem grape ferns is removed by activated carbon and a clarifier, wherein the amount of the activated carbon is 0.05-1% of the mass of the crude extract of long-stem grape ferns, and the amount of the clarifier is 1-5% of the mass of the crude extract of long-stem grape ferns.

8. The method for preparing the long-stem grape fern extract according to claim 7, characterized in that: In the step S3, cation exchange resin and anion exchange resin chromatography are used to perform desalination treatment.

9. A long-stem grape fern extract, characterized in that Prepared according to the preparation method according to any one of claims 1 to 8.

10. A use of the long-stem grape fern extract according to claim 9, characterized in that: The long-stem grape fern algae extract is used in the preparation of cosmetics.

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