A composition for anti-aging and firming skin and a method for preparing the same

Abstract

The application provides an anti-aging skin tightening composition and a preparation method thereof, and belongs to the technical field of anti-aging skin care, wherein the composition comprises a Haloxylon ammodendron fruit axis extract, a okra seed extract, tetrapeptide-4, acetyl octapeptide-1, a Rissoa parva extract, paeonol and a carnosine-zinc gluconate chelate. The Haloxylon ammodendron fruit axis powder is subjected to aerobic fermentation by Aspergillus oryzae, and the centrifugal supernatant is purified by a composite filler resin column to obtain the Haloxylon ammodendron fruit axis extract. The okra seed is subjected to sequential enzymolysis by composite enzymes, bromelin and subtilisin, and then extracted by adding anhydrous ethanol, and the centrifugal supernatant is purified by an LH-20 dextran gel column to obtain the okra seed extract. The Rissoa parva meat is subjected to sequential enzymolysis by trypsin and earthworm kinase, and the centrifugal supernatant of the enzymolysis solution is subjected to ultrafiltration to obtain a Rissoa parva extract with a molecular weight of less than 3kDa. The components are used in cooperation to achieve the balance between efficient anti-aging and low irritation through the synergistic effect of three pathways of "inhibiting collagen degradation, promoting collagen synthesis and resisting sugar soothing".

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-aging skin care, and particularly relates to an anti-aging skin tightening composition and a preparation method thereof. BACKGROUND

[0002] In the young population, skin aging is usually caused by factors such as ultraviolet radiation, environmental pollution, and staying up late, which activate free radicals, cause oxidative stress, damage cell DNA, and at the same time lead to overexpression of matrix metalloproteinases (MMPs), further degrading the structure of the dermis, and finally showing signs of aging such as wrinkles, sagging, decreased elasticity, and blurred contours.

[0003] Early anti-aging skin tightening products are limited to physical improvements, such as forming a film-like structure on the skin surface with macromolecular components such as collagen and hyaluronic acid, temporarily filling fine lines and improving skin tightening. The effect is short-lived and easily lost due to cleaning. Subsequently, the industry shifted to the development of chemical ingredients. Retinol and retinoid components became the mainstream because they can stimulate collagen synthesis. However, these components have poor stability, are easily affected by light and oxidation, and have strong skin irritation, which can cause redness and skin peeling in sensitive skin. At the same time, single components only target a single target of "collagen synthesis", making it difficult to address the complex aging process driven by multiple pathways such as oxidation, inflammation, and glycosylation, and the overall anti-aging effect is limited.

[0004] In recent years, the use of multi-target synergistic formulations has become a core trend: by combining retinol derivatives (promoting collagen synthesis), peptides (inhibiting MMP activity), ergothioneine (scavenging free radicals), and carnosine (anti-glycosylation) ingredients, the four major aging pathways of "synthesis reduction, degradation acceleration, oxidative damage, and glycosylation" are simultaneously blocked. The use of plant extracts has also become more widespread. Components such as ginsenosides and astragalus polysaccharides have become important components of natural anti-aging formulations due to their anti-oxidant, anti-inflammatory, and mild properties.

[0005] However, current anti-aging products still fail to meet user expectations, and some products still have issues such as poor effectiveness, allergies, and safety hazards. Good results are associated with poor safety, and good safety is associated with poor results, and there is always a contradiction between the two. Therefore, the development of anti-aging skin tightening products still needs to focus on balancing "efficiency", "mildness", and "safety", and improving both efficacy and safety to meet the needs of users. SUMMARY

[0006] In view of the problems of poor effect, slow effect, and difficulty in balancing high efficiency, mildness and safety of existing anti-aging skin tightening ingredients, the present application provides an anti-aging skin tightening composition and a preparation method thereof, which uses special methods to extract natural ingredients such as Haloxylon ammodendron inflorescence axis extract, okra seed extract, and montfortia extract, and uses them in combination with tetrapeptide-4, acetyl octapeptide-1, paeonol, and zinc carnosine-glucose chelate, so as to break through the limitation of traditional single action "anti-aging not comprehensive" through the synergistic effect of the three pathways of "inhibiting collagen degradation, promoting collagen synthesis, and anti-glycosylated soothing"; through "high purity of active ingredients + mild action mechanism", the balance between "high efficiency of anti-aging" and "low irritation" is realized, and it is suitable for various skin conditions such as sensitive skin. The specific technical scheme is as follows:

[0007] An anti-aging skin tightening composition, the composition comprises the following mass fractions of raw materials: 5-8 parts of Haloxylon ammodendron inflorescence axis extract, 3-5 parts of okra seed extract, 1.5-2 parts of tetrapeptide-4, 1-1.5 parts of acetyl octapeptide-1, 1-1.5 parts of montfortia extract, 0.1-0.2 parts of paeonol, and 0.5-1 parts of zinc carnosine-glucose chelate;

[0008] The preparation of the Haloxylon ammodendron inflorescence axis extract comprises: the Haloxylon ammodendron inflorescence axis powder is subjected to aerobic fermentation by Aspergillus oryzae, and the supernatant is purified by a composite filler resin column to obtain a product; the components of the composite filler include HPD750 macroporous adsorption resin and D101 macroporous adsorption resin;

[0009] The preparation of the okra seed extract comprises: the okra seed is subjected to sequential enzymolysis by a composite enzyme, bromelin, and subtilisin, anhydrous ethanol is added for extraction, and the supernatant is purified by an LH-20 dextran gel column to obtain a product; the composite enzyme includes cellulase and pectinase;

[0010] The preparation of the montfortia extract comprises: the montfortia meat is subjected to sequential enzymolysis by trypsin and earthworm kinase, the supernatant of the enzymolysis liquid is subjected to ultrafiltration to obtain a product with a molecular weight of less than 3kDa.

[0011] In the above-mentioned composition, the preparation method of the Haloxylon ammodendron inflorescence axis extract comprises: the Haloxylon ammodendron inflorescence axis is dried and then pulverized into powder, the powder is mixed with deionized water at a mass ratio of 1: (8-10), sterilized and cooled, and then trypsin and potassium dihydrogen phosphate are added, and Aspergillus oryzae activated bacteria liquid is added, and aerobic fermentation is carried out at 28-32 DEG C for 72-80h to obtain a fermentation liquid; centrifugation is carried out, the supernatant is filtered by a microfiltration membrane to remove bacteria, concentrated under reduced pressure, and then loaded onto a composite filler resin column, washed with 1.5-2 BV of 10-15% volume concentration ethanol aqueous solution to remove impurities, and then eluted with 3-4 BV of 60-65% volume concentration ethanol aqueous solution, the eluate is collected, concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the extract.

[0012] In the preparation method of the extract of the Elaeagnus pungens inflorescence axis, the particle size of the powder is 40-60 mesh; the sterilization is 121-125 DEG C for 15-20 min; the cooling is to 28-32 DEG C; the addition amount of the tryptone is 0.5-1% of the mass of the powder; the addition amount of the potassium dihydrogen phosphate is 0.05-0.1% of the mass of the powder; the bacterial content of the activated Aspergillus oryzae bacterial solution is 3*10 9 CFU / mL-6*10 9 CFU / mL; the addition amount of the activated Aspergillus oryzae bacterial solution is 2-4% of the mass of the powder; the centrifugation is 7000-9000 rpm for 15-20 min; the pore size of the microfiltration membrane is 0.22 mu m; the supernatant is concentrated to 20-25% of the volume under reduced pressure; the temperature of the concentration under reduced pressure is 45-50 DEG C; the components of the composite filler are (70-80):(20-30) mass ratio of HPD750 macroporous adsorption resin and D101 macroporous adsorption resin.

[0013] In the composition, the preparation method of the okra seed extract comprises the following steps: after the okra seed is dried, the okra seed is crushed into seed powder, the seed powder is mixed with deionized water at a mass ratio of 1:(10-15), the pH is adjusted to 4.5-5.0, a composite enzyme is added, and the enzyme hydrolysis is carried out at 50-55 DEG C for 2-3 h; the temperature is increased to 50-58 DEG C, the pH is adjusted to 6.5-7.0, ficin is added, and the enzyme hydrolysis is carried out for 1-1.5 h; the enzyme is inactivated; the temperature is decreased to 50-55 DEG C, the pH is adjusted to 7.0-7.5, subtilisin is added, and the enzyme hydrolysis is carried out for 1-1.5 h; the enzyme is inactivated, the temperature is decreased to room temperature, anhydrous ethanol is added, stirring extraction is carried out, centrifugation is carried out, the supernatant is taken, concentration is carried out under reduced pressure, the extract is loaded onto an LH-20 dextran gel column, 2-3 BV of deionized water is used for elution and impurity removal, and then 3-4 BV of 50-60% volume concentration ethanol aqueous solution is used for elution; the eluate is collected, the ethanol is removed under reduced pressure, and freeze-drying is carried out to obtain the extract.

[0014] In the preparation method of the okra seed extract, the particle size of the seed powder is 60-80 mesh; the composite enzyme is cellulase and pectinase at a mass ratio of 1:1; the addition amount of the composite enzyme is 2-3% of the mass of the seed powder; the addition amount of the ficin is 1-1.5% of the mass of the seed powder; the addition amount of the subtilisin is 1-1.5% of the mass of the seed powder; and the enzyme inactivation is carried out at 85-90 DEG C for 10-15 min.

[0015] In the preparation method of the okra seed extract, the amount of anhydrous ethanol added is 8 to 10 times the mass of the seed powder; the stirring extraction is carried out at 200 rpm to 300 rpm for 1 h to 1.5 h; the centrifugation is carried out at 4000 rpm to 5000 rpm for 15 min to 20 min; the supernatant is concentrated under reduced pressure to 20% to 25% of the volume; and the temperature of the concentration under reduced pressure is 45℃ to 50℃.

[0016] In the composition, the preparation method of the haminoaea cumingii extract includes the following steps: grinding and homogenizing haminoaea cumingii meat to obtain meat slurry, mixing the meat slurry with deionized water at a mass ratio of 1: (12-15), adjusting the pH to 8.0-8.5, adding trypsin, and carrying out enzymatic hydrolysis at 37℃-40℃ for 1.5 h-2 h, inactivating the enzyme, reducing the temperature to room temperature, adjusting the pH to 6.5-7.5, adding ascaricin, and carrying out enzymatic hydrolysis at 37℃-40℃ for 1.5 h-2 h, inactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, ultrafiltrating through a 3 kDa ultrafiltration membrane, taking a component with a molecular weight of less than 3 kDa, and freeze-drying to obtain the haminoaea cumingii extract.

[0017] In the preparation method of the haminoaea cumingii extract, the amount of trypsin added is 0.8%-1.2% of the mass of the meat slurry; the amount of ascaricin added is 0.5%-1.0% of the mass of the meat slurry; the inactivation of the enzyme is carried out at 85℃-90℃ for 10 min-15 min; and the centrifugation is carried out at 8000 rpm-8500 rpm for 10 min-15 min.

[0018] In the composition, the preparation method of the carnosine-zinc gluconate chelate includes the following steps: dissolving carnosine in 20-25 times the mass of deionized water at a mass ratio of carnosine: zinc gluconate = (1.5-1.8): 1 to obtain a carnosine solution; dissolving zinc gluconate in 15-20 times the mass of deionized water to obtain a zinc gluconate solution; under stirring, adding the zinc gluconate solution into the carnosine solution, adjusting the pH to 5.7-6.3, stirring at 30℃-35℃ for 1.5 h-2 h, concentrating under reduced pressure to obtain a concentrated solution, adding anhydrous ethanol, stirring to crystallize, and then filtering to obtain a filter cake, and freeze-drying to obtain the carnosine-zinc gluconate chelate.

[0019] In the preparation method of the carnosine-zinc gluconate chelate, the stirring speed is 300 rpm-400 rpm; the concentration under reduced pressure is carried out at 40℃-45℃ to 25%-30% of the volume; and the amount of anhydrous ethanol added is 3-4 times the volume of the concentrated solution.

[0020] The preparation method of the composition for anti-aging and skin tightening includes the following steps:

[0021] The composition is prepared by dispersing or dissolving, in a solvent, the extract of the fruiting axis of Kalidium foliatum, the extract of the seed of Abelmoschus esculentus, tetrapeptide-4, acetyl octapeptide-1, the extract of Facelinidae, paeonol, and zinc carnosine-glucose chelate in a mass ratio, and then mixing them.

[0022] The composition is compounded with pharmaceutically or cosmetically acceptable ingredients to prepare a product for anti-aging and skin tightening.

[0023] The composition and the preparation method thereof provided by the present application have the following beneficial effects:

[0024] Firstly, the composition of the present application breaks through the limitation of traditional single action "anti-aging is not comprehensive" through the synergy of the three pathways of "inhibiting collagen degradation, promoting collagen synthesis, and anti-glycation". The composition abandons irritating ingredients such as retinol and high-concentration acids, and all uses natural biological technology raw materials. Through "high-purity active ingredients + mild action mechanism", the composition realizes the balance between "high-efficiency anti-aging" and "low irritation", and is suitable for various skin conditions such as sensitive skin.

[0025] Secondly, the extract of the fruiting axis of Kalidium foliatum is subjected to aerobic fermentation of Aspergillus oryzae (decomposing cell walls to release active ingredients such as polyphenols, flavonoids, and proteins, without acidic metabolites to destroy the structure) + HPD750 and D101 composite resin purification (adsorbing active ingredients of different polarities and removing impurities), and has strong free radical scavenging (antioxidation) and MMP-1 inhibition (reducing collagen degradation), thereby providing the core support for anti-aging.

[0026] Thirdly, the extract of the seed of Abelmoschus esculentus is subjected to cellulase-pectinase (decomposing cell walls), ficin, and subtilisin (stepwise enzymatic hydrolysis to release effective active peptides) + LH-20 purification (separating active peptides from small molecular impurities), thereby assisting in enhancing the skin barrier repair ability and synergistically improving the anti-aging stability with other ingredients.

[0027] Fourthly, tetrapeptide-4 and acetyl octapeptide-1 act on fibroblasts to specifically promote collagen synthesis, and have mild and non-irritating effects, thereby providing a precise target for the "promoting synthesis" pathway.

[0028] Fifthly, the extract of Facelinidae is subjected to trypsin-ase and lysozyme double enzymolysis (fully decomposing proteins into small molecular active peptides) + 3kDa ultrafiltration (removing macromolecular impurities and retaining active components), and has good anti-aging effect, but has no obvious irritation, thereby solving the defects of traditional retinoids such as poor stability and strong irritation, and supplementing the mildness of the "promoting synthesis" pathway.

[0029] Sixthly, zinc carnosine-glucose chelate is chelated in a specific mass ratio (optimizing chelation efficiency and improving stability), specifically inhibits advanced glycation end products, reduces collagen cross-linking and skin relaxation, and enhances the efficacy of the "anti-glycation" pathway.

[0030] Seven, the ratio of each component of the composition of the present application has good synergistic effect: the extract of Kalidium foliatum, tetrapeptide-4, acetyl octapeptide-1 and the extract of Thais clavigera can perform "inhibition of degradation + promotion of synthesis" synergy, bidirectional action on collagen metabolic cycle, avoid the low efficiency problem of "only synthesis without protection" or "only protection without synthesis", realize the double optimization of collagen content and structure. The extract of Kalidium foliatum and the extract of Thais clavigera perform "antioxidation + mild anti-aging" complementary synergy, and together improve the resistance of the composition to aging.

[0031] The extract of Thais clavigera, paeonol and carnosine-zinc gluconate chelate perform "anti-aging + soothing anti-sugar" adaptive synergy, and the immediate soothing effect of paeonol can neutralize the slight irritation of the extract of Thais clavigera, realizing the dual goals of "efficacy adaptation to modern aging inducement + mildness guarantee". DETAILED DESCRIPTION

[0032] The present application will be further described below in combination with specific implementation cases, but the present application is not limited to these examples.

[0033] Example 1

[0034] An anti-aging and skin tightening composition, comprising the following raw materials in mass fraction: 6.5 parts of Kalidium foliatum fruit axis extract, 4 parts of abelmoschus esculentus seed extract, 1.8 parts of tetrapeptide-4, 1.3 parts of acetyl octapeptide-1, 1.2 parts of Thais clavigera extract, 0.15 parts of paeonol, 0.8 parts of carnosine-zinc gluconate chelate.

[0035] The preparation method of the Kalidium foliatum fruit axis extract comprises:

[0036] Strain activation: the Aspergillus oryzae is activated and cultured with PDA liquid medium to obtain an activated Aspergillus oryzae bacterial solution with a bacterial content of 4x10 9 CFU / mL.

[0037] Fermentation extraction: after the Kalidium foliatum fruit axis is dried and crushed through a 50-mesh sieve, powder is obtained, the powder is mixed with deionized water at a mass ratio of 1:9, sterilized at 121℃ for 18 min, and cooled to 30℃; 0.8% of tryptone, 0.06% of potassium dihydrogen phosphate and 3% of the activated Aspergillus oryzae bacterial solution are added to the powder, and aerobic fermentation is carried out at 30℃ for 76 h to obtain a fermentation liquid.

[0038] Purification: centrifugation at 8000 rpm for 18 min, and the supernatant was filtered through a 0.22 μm microfiltration membrane to remove bacteria; concentrated to 23% of the original volume at 48°C under reduced pressure, and then loaded onto a composite filler resin column (HPD750 macroporous adsorption resin:D101 macroporous adsorption resin=75:25 mass ratio, soaked with 92% volume concentration ethanol aqueous solution for 25 h, and then eluted with anhydrous ethanol until the effluent was not white turbid after adding water, and then washed with deionized water to remove ethanol), and then eluted with 1.5 BV of 12% volume concentration ethanol aqueous solution to remove impurities, and then eluted with 3.5 BV of 62% volume concentration ethanol aqueous solution, and then collected the eluate, removed ethanol at 48°C under reduced pressure, and then freeze-dried to obtain the extract of the fruiting branch of Haloxylon ammodendron.

[0039] The preparation method of the okra seed extract comprises the following steps:

[0040] Enzymolysis: after the okra seeds are dried and crushed to break the shells, the seed powder is obtained by passing through a 60-mesh sieve, mixed with deionized water at a mass ratio of 1:12, the pH is adjusted to 4.8, and 2.5% of the mass of the seed powder of a composite enzyme (cellulase:pectinase=1.3:1) is added, and then the seed powder is enzymolyzed at 52°C for 2.5 h; the temperature is increased to 55°C, the pH is adjusted to 6.8, 1.2% of the mass of the seed powder of ficin is added, and then the seed powder is enzymolyzed for 1 h, and then the enzyme is inactivated at 88°C for 12 min; the temperature is decreased to 53°C, the pH is adjusted to 7.2, 1.3% of the mass of the seed powder of subtilisin is added, and then the seed powder is enzymolyzed for 1 h, and then the enzyme is inactivated at 88°C for 12 min, and then the temperature is decreased to room temperature.

[0041] Extraction and purification: 9 times the mass of the seed powder of anhydrous ethanol is added, and then the mixture is stirred at 250 rpm for 1 h, and then centrifuged at 4500 rpm for 18 min, and then the supernatant is taken, and then concentrated to 22% of the volume at 48°C under reduced pressure, and then loaded onto an LH-20 dextran gel column, and then eluted with 2.5 BV of deionized water to remove impurities, and then eluted with 3.5 BV of 55% volume concentration ethanol aqueous solution, and then the eluate is collected, and then ethanol is removed at 48°C under reduced pressure, and then freeze-dried to obtain the okra seed extract.

[0042] The preparation method of the mountain snail extract comprises the following steps:

[0043] Enzymolysis: the washed mountain snail meat is crushed and homogenized to obtain snail slurry, mixed with deionized water at a mass ratio of 1:13, the pH is adjusted to 8.3, and then 1.0% of the mass of the snail slurry of trypsin is added, and then the snail slurry is enzymolyzed at 37°C for 1.5 h, and then the enzyme is inactivated at 88°C for 12 min, and then the temperature is decreased to room temperature, the pH is adjusted to 7.0, and then 0.8% of the mass of the snail slurry of ascaricin is added, and then the snail slurry is enzymolyzed at 37°C for 1.5 h, and then the enzyme is inactivated at 88°C for 12 min, and then the temperature is decreased to room temperature to obtain an enzymolysis solution.

[0044] Purification: the enzymolysis solution is centrifuged at 8200 rpm for 12 min, and then the supernatant is taken, and then ultrafiltrated through a 3-kDa ultrafiltration membrane, and then the components below 3 kDa are taken, and then freeze-dried to obtain the mountain snail extract.

[0045] The preparation method of the carnosine-zinc gluconate chelate comprises the following steps:

[0046] Liquid preparation: Carnosine is dissolved in 23 times the mass of deionized water according to a mass ratio of carnosine: zinc gluconate = 1.6:1 to obtain a carnosine solution; zinc gluconate is dissolved in 18 times the mass of deionized water to obtain a zinc gluconate solution.

[0047] Chelation: The zinc gluconate solution is added to the carnosine solution under stirring at 350 rpm, the pH is adjusted to 6.0, and stirring is performed at 32°C and 350 rpm for 1.5 h; the concentrated solution is concentrated to 28% of the volume under reduced pressure at 43°C to obtain a concentrated solution; 3.5 times the volume of anhydrous ethanol is added to the concentrated solution, and crystallization is performed under stirring; the filter cake is obtained after suction filtration, and is freeze-dried to obtain the carnosine-zinc gluconate chelate.

[0048] Example 2

[0049] An anti-aging and skin tightening composition comprises the following raw materials in mass parts: 5 parts of Haloxylon ammodendron fruit axis extract, 5 parts of Abelmoschus esculentus seed extract, 1.5 parts of tetrapeptide-4, 1.5 parts of acetyl octapeptide-1, 1 part of Thais clavigera extract, 0.2 parts of paeonol, and 0.5 parts of carnosine-zinc gluconate chelate.

[0050] The preparation method of the Haloxylon ammodendron fruit axis extract comprises the following steps:

[0051] Strain activation: Aspergillus oryzae is activated and cultured in a PDA liquid medium to obtain an activated Aspergillus oryzae bacterial solution with a bacterial content of 3×10 9 CFU / mL.

[0052] Fermentation extraction: After the Haloxylon ammodendron fruit axis is dried, it is crushed through a 60-mesh sieve to obtain a powder; the powder is mixed with deionized water according to a mass ratio of 1:8, sterilized at 123°C for 20 min, and cooled to 28°C; 1% of the mass of the powder is added as tryptone, 0.05% of the mass of the powder is added as potassium dihydrogen phosphate, and 4% of the mass of the powder is added as the activated Aspergillus oryzae bacterial solution; aerobic fermentation is performed at 28°C for 72 h to obtain a fermentation liquid.

[0053] Purification: The fermentation liquid is centrifuged at 7000 rpm for 20 min, and the supernatant is filtered through a 0.22-μm microfiltration membrane to remove bacteria; the supernatant is concentrated to 25% of the original volume under reduced pressure at 50°C; the concentrated solution is loaded onto a composite filler resin column (HPD750 macroporous adsorption resin:D101 macroporous adsorption resin = 80:20 in mass ratio, which is swelled by being soaked in 95% volume concentration ethanol aqueous solution for 24 h, and then is eluted with anhydrous ethanol until the eluent does not become white and turbid after being added with water; after the column is washed with deionized water to remove ethanol, the column is used); the column is eluted with 1.5 BV of 15% volume concentration ethanol aqueous solution to remove impurities, and then is eluted with 3 BV of 65% volume concentration ethanol aqueous solution; the eluate is collected, concentrated to remove ethanol under reduced pressure at 50°C, and freeze-dried to obtain the Haloxylon ammodendron fruit axis extract.

[0054] The preparation method of the okra seed extract comprises:

[0055] Enzymolysis: after drying, the okra seeds are crushed and broken to pass through an 80-mesh sieve to obtain seed powder, which is mixed with deionized water at a mass ratio of 1:10, the pH is adjusted to 5.0, 2% of the compound enzyme (cellulase: pectinase = 1.5:1) of the mass of the seed powder is added, and the enzyme is hydrolyzed at 50℃ for 3h; the temperature is raised to 50℃, the pH is adjusted to 7.0, 1% of the bromelain of the mass of the seed powder is added, and the enzyme is hydrolyzed for 1.5h, and the enzyme is inactivated at 85℃ for 15min; the temperature is lowered to 50℃, the pH is adjusted to 7.0, 1.5% of the bacillus subtilis protease of the mass of the seed powder is added, and the enzyme is hydrolyzed for 1h, and the enzyme is inactivated at 90℃ for 10min, and then the temperature is lowered to room temperature.

[0056] Extraction and purification: 10 times the mass of the seed powder of anhydrous ethanol is added, stirring extraction is performed at 200rpm for 1.5h, centrifugation is performed at 4000rpm for 20min, the supernatant is taken, and the volume is concentrated to 25% at 50℃ under reduced pressure; the sample is loaded into an LH-20 dextran gel column, 2BV of deionized water is used for elution and impurity removal, and then 4BV of 50% volume concentration ethanol aqueous solution is used for elution; the eluate is collected, the ethanol is removed at 50℃ under reduced pressure, and freeze-drying is performed to obtain the okra seed extract.

[0057] The preparation method of the okra seed extract comprises:

[0058] Enzymolysis: the washed mountain knot snail meat is pulverized and homogenized to obtain snail slurry, which is mixed with deionized water at a mass ratio of 1:12, the pH is adjusted to 8.0, 0.8% of the trypsin of the mass of the snail slurry is added, and the enzyme is hydrolyzed at 38℃ for 1.5h, the enzyme is inactivated at 90℃ for 10min, the temperature is lowered to room temperature, the pH is adjusted to 6.5, 0.5% of the avenin of the mass of the snail slurry is added, and the enzyme is hydrolyzed at 38℃ for 1.5h, the enzyme is inactivated at 90℃ for 10min, and then the temperature is lowered to room temperature to obtain an enzyme hydrolysate.

[0059] Purification: the enzyme hydrolysate is centrifuged at 8000rpm for 10min, the supernatant is taken, ultrafiltration is performed on a 3kDa ultrafiltration membrane, the components below 3kDa are taken, and freeze-drying is performed to obtain the mountain knot snail extract.

[0060] The preparation method of the carnosine-zinc gluconate chelate comprises:

[0061] Liquid preparation: according to the mass ratio of carnosine: zinc gluconate = 1.5:1, the carnosine is dissolved in 25 times the mass of deionized water to obtain a carnosine solution; and the zinc gluconate is dissolved in 20 times the mass of deionized water to obtain a zinc gluconate solution.

[0062] Chelation: under the condition of stirring at 300 rpm, the zinc gluconate solution was added into the carnosine solution, the pH was adjusted to 5.7, and the mixture was stirred at 30 ℃ for 1.5 h. The mixture was concentrated to 25% of the original volume at 45 ℃ under reduced pressure to obtain a concentrated solution. The concentrated solution was added with 4 times the volume of anhydrous ethanol, and the mixture was stirred to precipitate crystals. After suction filtration, the filter cake was obtained, and was freeze-dried to obtain the carnosine-zinc gluconate chelate.

[0063] Example 3

[0064] A composition for anti-aging and firming skin, comprising the following raw materials in mass parts: 8 parts of Haloxylon ammodendron fruit axis extract, 3 parts of Abelmoschus esculentus seed extract, 2 parts of tetrapeptide-4, 1 part of acetyl octapeptide-1, 1.5 parts of Thais clavigera extract, 0.1 part of paeonol, and 1 part of carnosine-zinc gluconate chelate.

[0065] The preparation method of the Haloxylon ammodendron fruit axis extract comprises the following steps:

[0066] Activation of the strain: the Aspergillus oryzae was activated and cultured with PDA liquid medium to obtain an activated Aspergillus oryzae bacterial solution with a bacterial content of 6×10 9 CFU / mL.

[0067] Fermentation and extraction: after the Haloxylon ammodendron fruit axis was dried, the dried fruit axis was ground through a 40-mesh sieve to obtain a powder. The powder was mixed with deionized water at a mass ratio of 1:10, sterilized at 125 ℃ for 15 min, and cooled to 32 ℃. Then, 0.5% of tryptone, 0.1% of potassium dihydrogen phosphate, and 2% of the activated Aspergillus oryzae bacterial solution were added to the powder, and the mixture was subjected to aerobic fermentation at 32 ℃ for 80 h to obtain a fermentation liquor.

[0068] Purification: the fermentation liquor was centrifuged at 9000 rpm for 15 min, and the supernatant was filtered through a 0.22-μm microfiltration membrane to remove bacteria. Then, the supernatant was concentrated to 20% of the original volume at 45 ℃ under reduced pressure. The concentrated solution was loaded onto a composite filler resin column (HPD750 macroporous adsorption resin:D101 macroporous adsorption resin=70:30 in mass ratio, which was swelled by being soaked in 90% volume concentration ethanol aqueous solution for 26 h, and then washed with anhydrous ethanol until the water added to the effluent did not become white and turbid, and then washed with deionized water to remove ethanol). The column was washed with 2 BV of 10% volume concentration ethanol aqueous solution to remove impurities, and then eluted with 4 BV of 60% volume concentration ethanol aqueous solution. The eluate was collected, concentrated to remove ethanol at 45 ℃ under reduced pressure, and freeze-dried to obtain the Haloxylon ammodendron fruit axis extract.

[0069] The preparation method of the Abelmoschus esculentus seed extract comprises the following steps:

[0070] Enzymatic hydrolysis: The dried okra seeds were crushed and broken into pieces, and then passed through a 60-mesh sieve to obtain seed powder. The seed powder was mixed with deionized water at a mass ratio of 1:15, the pH was adjusted to 4.5, and 3% of the mass of the seed powder was added with a compound enzyme (cellulase: pectinase = 1:1). Enzymatic hydrolysis was performed at 55°C for 2 hours. The temperature was increased to 58°C, the pH was adjusted to 6.5, and 1.5% of the mass of the seed powder was added with ficin. Enzymatic hydrolysis was performed for 1 hour, and the enzyme was inactivated at 90°C for 10 minutes. The temperature was decreased to 55°C, the pH was adjusted to 7.5, and 1% of the mass of the seed powder was added with subtilisin. Enzymatic hydrolysis was performed for 1.5 hours, the enzyme was inactivated at 85°C for 15 minutes, and the temperature was decreased to room temperature.

[0071] Extraction and purification: 8 times the mass of the seed powder was added with anhydrous ethanol, and extraction was performed at 300 rpm for 1 hour. Centrifugation was performed at 5000 rpm for 15 minutes, and the supernatant was collected. Concentration was performed at 45°C under reduced pressure to 20% of the volume. The sample was loaded onto an LH-20 dextran gel column, and impurities were removed by elution with 3 BV of deionized water. Elution was performed with 3 BV of 60% volume concentration of an ethanol aqueous solution. The eluate was collected, and ethanol was removed by concentration at 45°C under reduced pressure. Freeze-drying was performed to obtain an okra seed extract.

[0072] The preparation method of the mountain snail extract includes:

[0073] Enzymatic hydrolysis: The washed mountain snail meat was crushed and homogenized to obtain snail slurry. The snail slurry was mixed with deionized water at a mass ratio of 1:15, the pH was adjusted to 8.5, and 1.2% of the mass of the snail slurry was added with trypsin. Enzymatic hydrolysis was performed at 40°C for 2 hours. The enzyme was inactivated at 85°C for 15 minutes, and the temperature was decreased to room temperature. The pH was adjusted to 7.5, and 1.0% of the mass of the snail slurry was added with an earthworm kinase. Enzymatic hydrolysis was performed at 40°C for 2 hours. The enzyme was inactivated at 85°C for 15 minutes, and the temperature was decreased to room temperature to obtain an enzymatic hydrolysate.

[0074] Purification: The enzymatic hydrolysate was centrifuged at 8500 rpm for 15 minutes, and the supernatant was collected. Ultrafiltration was performed with a 3-kDa ultrafiltration membrane, and components below 3 kDa were collected. Freeze-drying was performed to obtain a mountain snail extract.

[0075] The preparation method of the carnosine-zinc gluconate chelate includes:

[0076] Liquid preparation: Carnosine was dissolved in 20 times the mass of deionized water at a mass ratio of carnosine: zinc gluconate = 1.8:1 to obtain a carnosine solution. Zinc gluconate was dissolved in 15 times the mass of deionized water to obtain a zinc gluconate solution.

[0077] Chelation: The zinc gluconate solution was added to the carnosine solution under stirring at 400 rpm, the pH was adjusted to 6.3, and stirring was performed at 35°C and 400 rpm for 2 hours. Concentration was performed at 40°C under reduced pressure to 30% of the volume to obtain a concentrated solution. Anhydrous ethanol was added to the concentrated solution at 3 times the volume, and crystallization was performed under stirring. Filtration was performed to obtain a filter cake, and freeze-drying was performed to obtain a carnosine-zinc gluconate chelate.

[0078] A method for preparing an anti-aging skin tightening composition according to each of the above embodiments comprises the following steps:

[0079] The Alhagi sparsifolia Franch. fruit stalk extract, the Abelmoschus esculentus seed extract, the tetrapeptide-4, the acetyl octapeptide-1, the Acrotheca monticola extract, the paeonol, and the carnosine-zinc gluconate are dispersed or dissolved in a solvent according to the mass fraction in each embodiment, and then mixed to obtain the composition.

[0080] The anti-aging skin tightening composition according to each of the above embodiments is compounded with a pharmaceutically or cosmetically acceptable ingredient to prepare a product for anti-aging skin tightening.

[0081] Comparative Example 1

[0082] The Alhagi sparsifolia Franch. fruit stalk extract is modified to 9 parts, and the Abelmoschus esculentus seed extract is modified to 1.5 parts; other parameters and methods are the same as in Example 1.

[0083] Comparative Example 2

[0084] The Alhagi sparsifolia Franch. fruit stalk extract is modified to 1.2 parts, and the Acrotheca monticola extract is modified to 6.5 parts; other parameters and methods are the same as in Example 1.

[0085] Comparative Example 3

[0086] The Abelmoschus esculentus seed extract is modified to 1 part, and the Acrotheca monticola extract is modified to 4.2 parts; other parameters and methods are the same as in Example 1.

[0087] Comparative Example 4

[0088] The Acrotheca monticola extract is modified to 0.5 parts, and the carnosine-zinc gluconate chelate is modified to 1.5 parts; other parameters and methods are the same as in Example 1.

[0089] Comparative Example 5

[0090] In the preparation of the Alhagi sparsifolia Franch. fruit stalk extract, Aspergillus oryzae is replaced by Lactobacillus plantarum (anaerobic fermentation); other parameters and methods are the same as in Example 1.

[0091] Comparative Example 6

[0092] In the preparation of the Alhagi sparsifolia Franch. fruit stalk extract, the resin column is only filled with HPD750 macroporous adsorption resin; other parameters and methods are the same as in Example 1.

[0093] Comparative Example 7

[0094] In the preparation of the Alhagi sparsifolia Franch. fruit stalk extract, the resin column is only filled with D101 macroporous adsorption resin; other parameters and methods are the same as in Example 1.

[0095] Comparative Example 8

[0096] In the preparation of the extract of the inflorescence axis of Tribulus terrester L., the HPD750 macroporous adsorption resin is replaced by AB-8 macroporous adsorption resin; other parameters and methods are the same as in Example 1.

[0097] Comparative Example 9

[0098] In the preparation of the extract of the seed of Abelmoschus esculentus L., the ficin is replaced by bromelin; other parameters and methods are the same as in Example 1.

[0099] Comparative Example 10

[0100] In the preparation of the extract of the seed of Abelmoschus esculentus L., the subtilisin is replaced by papain; other parameters and methods are the same as in Example 1.

[0101] Comparative Example 11

[0102] In the preparation of the extract of the seed of Abelmoschus esculentus L., the subtilisin is not used for enzymolysis; other parameters and methods are the same as in Example 1.

[0103] Comparative Example 12

[0104] In the preparation of the extract of the seed of Abelmoschus esculentus L., the LH-20 Sephadex gel column is replaced by G-25 Sephadex gel; other parameters and methods are the same as in Example 1.

[0105] Comparative Example 13

[0106] In the preparation of the extract of the seed of Anguiskeenia philippinensis, the lumbrokinase is replaced by bromelin; other parameters and methods are the same as in Example 1.

[0107] Comparative Example 14

[0108] In the preparation of the extract of the seed of Anguiskeenia philippinensis, the trypsin is not used for enzymolysis; other parameters and methods are the same as in Example 1.

[0109] The raw material sources involved in the above examples and comparative examples: Tetrapeptide-4 was from Chengdu Yunxi Chemical Co., Ltd. Acetyl octapeptide-1 was from Chengdu Kecheng Fine Chemical Co., Ltd. Paeonol was from Jiangxi Hairei Natural Plant Co., Ltd. Aspergillus oryzae was from Koland Biotech (Shanghai) Co., Ltd., product number BNCC 142787. Trypsin was from Jinan Huirui Biotechnology Co., Ltd., from YD-02. Cellulase was from Shandong Yaqiu Biotechnology Co., Ltd., enzyme activity 100,000 U / g. Pectinase was from Wuhan Kairan Biotechnology Co., Ltd., enzyme activity 30,000 U / g. Ficus protease was from Nanjing Songguan Biotechnology Co., Ltd., enzyme activity 100,000 U / g. Subtilisin was from Wuhan Shuer Biotechnology Co., Ltd., enzyme activity 100,000 U / g. HPD750 macroporous adsorption resin was from Shanghai Yuanye Biotechnology Co., Ltd., model S27400. D101 macroporous adsorption resin was from Shanghai Yuanye Biotechnology Co., Ltd., model S14161. LH-20 dextran gel was from Shanghai Yuanye Biotechnology Co., Ltd., model S14037. Carnosine was from Hangzhou Weipulai Biotechnology Co., Ltd., L-carnosine. Zinc gluconate was from Shaanxi Mingchong Pharmaceutical Co., Ltd., pharmaceutical grade. Trypsin was from Heilongjiang Beiheli Biotechnology Co., Ltd., enzyme activity 200,000 U / g. Aelitase was from Wuhan Xinxinjiali Biotechnology Co., Ltd., enzyme activity 20,000 iu / mg. Papain was from Xi'an Komprise Biotechnology Co., Ltd., enzyme activity 100,000 U / g. Bromelain was from Peizuo Biotechnology (Xi'an) Co., Ltd., enzyme activity 100,000 U / g. AB-8 macroporous adsorption resin was from Shanghai Yuanye Biotechnology Co., Ltd., model S30931. G-25 dextran gel was from Shanghai Yuanye Biotechnology Co., Ltd., model S14031. Lactobacillus plantarum was from Koland Biotech (Shanghai) Co., Ltd., BNCC 138404.

[0110] I. Cytotoxicity test:

[0111] Sample preparation: The composition powder was diluted with DMEM culture solution to a test concentration of 250 μg / mL, and filtered through a 0.22 μm filter membrane to remove bacteria.

[0112] The experimental steps include: HaCaT (human immortal keratinocytes) and HDF cells (human dermal fibroblasts) were subjected to proliferation rate test respectively. The cells were cultured in DMEM medium (containing 10% FBS, 1% penicillin-streptomycin) at 37°C, 5% CO2 to logarithmic growth phase. The cells were seeded at 5×10 3Cells were seeded at a density of 100 μL of culture medium per well in 96-well plates and cultured for 24 hours to allow for full cell adhesion. The old culture medium was discarded and replaced with 100 μL of fresh culture medium containing 100 μg / mL of the sample per well. Six replicates were set up for each group, including a blank control group (DMEM medium only) and a cell-based control group (DMEM medium + cells, no composite material). The cells were cultured at 37°C and 5% CO2 for another 24 hours. 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0113] Evaluation index: Cell viability (%) = (OD sample group - OD blank control group) / (OD cell baseline control group - OD blank control group) × 100%.

[0114] II. Detection of Collagen Synthesis Promotion Effect:

[0115] Sample preparation: The composition powder was diluted with DMEM culture medium to prepare a sample solution with a concentration of 250 μg / mL, and then filtered through a 0.22 μm filter membrane for sterilization.

[0116] The experimental procedure included: culturing HDF cells (human dermal fibroblasts); digesting and resuspending the cells when the cell confluence reached 85% or higher; and then loading the cells at 5 × 10⁶ cells / day. 4 Cells were seeded at a density of 100 cells / well in 24-well plates, with 1 mL of culture medium per well, and cultured for 24 hours. The old culture medium was discarded, and 1 mL of sample solution was added. Three parallel wells were set up for each concentration. A blank control group (containing only DMEM culture medium, without the composite) was also set up. The cells were cultured for another 48 hours. The cell culture supernatant was collected, and the type I collagen content was detected using an ELISA method: Following the ELISA kit instructions, standards and the test culture medium were added, incubated, washed, and then enzyme-labeled antibody was added. After incubation and washing, the substrate was added for color development, and the absorbance was measured at 450 nm. The collagen concentration was calculated based on the standard curve.

[0117] Evaluation index: Collagen synthesis promotion rate (%) = (Collagen content of sample group - Collagen content of blank group) / Collagen content of blank group × 100%.

[0118] III. Free radical scavenging ability test:

[0119] Sample preparation: The composition was diluted with anhydrous ethanol to prepare a sample solution with a concentration of 250 μg / mL.

[0120] The experimental steps include: preparing 0.1 mmol / L DPPH ethanol solution, and storing in the dark (use within 24 h after preparation). Take 2 mL of sample solution in a test tube, add 2 mL of DPPH ethanol solution, mix thoroughly, and react at room temperature for 30 min in the dark. Take anhydrous ethanol as a blank control to adjust zero, and measure the absorbance value at a wavelength of 517 nm:

[0121] Determination group absorbance: sample solution + DPPH solution. Sample blank group absorbance: sample solution + equal volume of anhydrous ethanol (used to correct the color of the sample solution itself). DPPH blank group absorbance: DPPH solution + equal volume of anhydrous ethanol (initial value of DPPH). Set three parallel samples.

[0122] Evaluation index: DPPH free radical scavenging rate (%) = [1- (determination group absorbance-sample blank group absorbance) / DPPH blank group absorbance] x 100%.

[0123] Four, matrix metalloproteinase (MMP-1) inhibition detection:

[0124] Sample preparation: dilute the composition with Tris-HCl buffer (pH 7.5) to a concentration of 250 μg / mL of sample solution, and filter sterilization through a 0.22 μm filter.

[0125] The experimental steps include: in a 96-well black plate, sequentially add 50 μL of MMP-1 enzyme solution (concentration 0.1 U / mL), 50 μL of sample solution, mix well, and pre-incubate at 37°C for 15 min; then add 200 μL of 10 μM fluorescent substrate, and mix gently. Incubate at 37°C in the dark for 60 min, and measure the fluorescence intensity (excitation wavelength 328 nm, emission wavelength 393 nm) using a fluorescence microplate reader. Blank control group: 50 μL of Tris-HCl buffer + 50 μL of Tris-HCl buffer + 100 μL of fluorescent substrate (correct background fluorescence). Sample background control group: 50 μL of Tris-HCl buffer + 50 μL of sample solution + 100 μL of fluorescent substrate. Negative control group: 50 μL of MMP-1 enzyme solution + 50 μL of Tris-HCl buffer + 100 μL of fluorescent substrate. Set three parallel samples.

[0126] Evaluation index: MMP-1 inhibition rate (%) = [1- (sample group fluorescence intensity-sample background control group fluorescence intensity) / (negative control group fluorescence intensity-blank control group fluorescence intensity)] x 100%.

[0127] Five, anti-glycosylation activity detection:

[0128] Sample preparation: dilute the composition with PBS buffer (pH 7.4) to a concentration of 250 μg / mL of sample solution, and filter sterilization through a 0.22 μm filter.

[0129] The experimental steps include: adding each component in the centrifuge tube according to the system shown in Table 1 (3 parallel samples are set), and adding PBS buffer to a total volume of 5 mL, mixing gently and sealing. Incubate at 37°C for 21 days in the dark to obtain the incubated reaction solution. Take 200 μL of the incubated reaction solution and use a fluorescence spectrophotometer to measure the fluorescence intensity (excitation wavelength 370 nm, emission wavelength 440 nm).

[0130] Table 1 Anti-glycosylation reaction system

[0131]

[0132] Evaluation index: AGEs inhibition rate (%) = [1- (fluorescence intensity of sample group-fluorescence intensity of sample background control group) / (fluorescence intensity of negative control group-fluorescence intensity of blank control group)] x 100%.

[0133] Table 2 Test results (average value)

[0134]

[0135] From the above results, it can be seen that the composition core of Examples 1 to 3 is: the formula proportion is adapted to the synergistic pathway: strictly follow the design of "inhibition of degradation + promotion of synthesis + anti-sugar relief", the proportion of each component is reasonable, the synergistic effect is good, and there is no pathway rupture or cytotoxicity caused by excess or deficiency. The milk vetch is fermented by Aspergillus oryzae + purified by composite resin, the okra seeds are subjected to three-step enzymatic hydrolysis + LH-20 purification, the mountain knot snails are subjected to double enzymatic hydrolysis + 3 kDa ultrafiltration, and the carnosine-zinc gluconate is chelated in proportion, the optimal extraction and purification scheme is designed, and the effective active ingredient content is high and the impurities are few.

[0136] In Comparative Examples 1 to 4, the proportion of each component is unbalanced, the synergistic effect of each component is reduced, leading to varying degrees of data deterioration, and excessive components will increase their own irritability and the toxicity of impurities, breaking the balance of "high efficiency-mildness".

[0137] The Aspergillus oryzae was replaced by Lactobacillus plantarum in the preparation of the extract of the fruiting branch axis of Elaeagnus pungens Bunge of Comparative Example 5. As a filamentous fungus, Aspergillus oryzae can efficiently decompose the cell wall of the fruiting branch axis of Elaeagnus pungens Bunge to release active ingredients such as polyphenols, flavonoids and active polypeptides, and the fermentation product is free of acidic substances, which will not damage the structure of the active ingredients. The main metabolic product of Lactobacillus plantarum is lactic acid, which can reduce the pH value of the fermentation system, resulting in the loss of the antioxidant activity of some components and the difference in the active polypeptides obtained. It cannot fully decompose macromolecular substances, and the yield of active ingredients is low. The antioxidant (DPPH clearance rate), collagen degradation inhibition (MMP-1 inhibition rate), AGEs inhibition rate and collagen synthesis promotion rate of the extract all decrease, and the residual impurities also reduce the cell survival rate.

[0138] The extract of the fruiting branch axis of Elaeagnus pungens Bunge of Comparative Example 6 and Comparative Example 7 is purified only by one kind of resin, which lacks the synergistic purification effect, the types of active ingredients obtained are reduced, and the types of some impurities are increased, which affects the overall effect.

[0139] In the purification of the extract of the fruiting branch axis of Elaeagnus pungens Bunge of Comparative Example 8, HPD750 is replaced by AB-8 macroporous adsorption resin. The pore size distribution and surface chemical properties of HPD750 and AB-8 are significantly different: the pore size of AB-8 is smaller, and some macromolecular polyphenols cannot enter the pore, and the surface has fewer polar groups and weaker hydrogen bonding ability, which can easily lead to the loss of active ingredients and the stimulation of cells by impurities. All indicators decrease.

[0140] Comparative Example 9 to Comparative Example 11: The replacement or absence of protease, different proteases and different enzyme combination and enzyme sequence strategies all affect the final peptide composition. The types of polypeptide chains are different, and the efficacy and stimulation are different, resulting in changes in the data.

[0141] In the purification of the extract of the okra seed of Comparative Example 12, LH-20 is replaced by G-25 Sephadex. The separation range of LH-20 matches the active ingredients of the okra seed extract, which can effectively separate the active ingredients and small molecular impurities. The separation range of G-25 cannot retain small molecular active peptides and cannot effectively remove small molecular impurities, and it also has weak adsorption capacity for macromolecular polysaccharides with a molecular weight of > 5000 Da, which leads to the loss of active ingredients and the residual impurities. The impurities can interfere with the binding of the active ingredients to the cell targets, and the absence of macromolecular polysaccharides weakens the skin barrier repair ability, which together leads to the decrease of all anti-aging indicators; the osmotic pressure of the impurities also reduces the cell survival rate.

[0142] In the preparation of the extract of the mountain knot snail of Comparative Example 13 and Comparative Example 14, the eelase is replaced by bromelain or the trypsin enzyme is omitted. Different proteases and different enzyme combination and enzyme sequence strategies all affect the final peptide composition. The types of polypeptide chains are different, and the efficacy and stimulation are different, resulting in changes in the data.

Claims

1. An anti-aging skin tightening composition, characterized in that, The composition comprises the following raw materials in mass fraction: 5-8 parts of Haloxylon ammodendron inflorescence axis extract, 3-5 parts of Abelmoschus esculentus seed extract, 1.5-2 parts of tetrapeptide-4, 1-1.5 parts of acetyl octapeptide-1, 1-1.5 parts of Facelina fusca extract, 0.1-0.2 parts of paeonol and 0.5-1 part of carnosine-zinc gluconate chelate; The preparation of the Haloxylon ammodendron inflorescence axis extract comprises: aerobic fermentation of Haloxylon ammodendron inflorescence axis powder by Aspergillus oryzae, centrifugation of the supernatant and purification of the supernatant by a composite filler resin column to obtain the product; the components of the composite filler include HPD750 macroporous adsorption resin and D101 macroporous adsorption resin; The preparation of the Abelmoschus esculentus seed extract comprises: sequential enzymolysis of Abelmoschus esculentus seeds by a composite enzyme, ficin and subtilisin, addition of anhydrous ethanol for extraction, centrifugation of the supernatant and purification of the supernatant by an LH-20 dextran gel column to obtain the product; the composite enzyme comprises cellulase and pectinase; The preparation of the Facelina fusca extract comprises: sequential enzymolysis of Facelina fusca meat by trypsin and levanidase, centrifugation of the supernatant of the enzymolysis solution and ultrafiltration to obtain a product with a molecular weight of less than 3kDa.

2. The skin anti-aging and firming composition according to claim 1, wherein The preparation method of the Haloxylon ammodendron inflorescence axis extract comprises: drying of Haloxylon ammodendron inflorescence axis, pulverization into powder, mixing of the powder with deionized water at a mass ratio of 1: (8-10), sterilization and cooling, addition of tryptone and potassium dihydrogen phosphate, addition of activated Aspergillus oryzae bacterial solution, aerobic fermentation at 28-32℃ for 72-80h to obtain a fermentation liquor; centrifugation, sterilization of the supernatant by microfiltration membrane, concentration under reduced pressure, loading onto a composite filler resin column, elution of impurities by 1.5-2BV of 10-15% volume concentration ethanol aqueous solution, further elution by 3-4BV of 60-65% volume concentration ethanol aqueous solution, collection of the eluate, removal of ethanol under reduced pressure, freeze-drying to obtain the extract.

3. The skin anti-aging and firming composition according to claim 2, wherein The particle size of the powder is 40-60 mesh; the sterilization is 121-125℃ for 15-20 minutes; the cooling is to 28-32℃; the added amount of the tryptone is 0.5-1% of the mass of the powder; the added amount of the potassium dihydrogen phosphate is 0.05-0.1% of the mass of the powder; the bacterial content of the activated Aspergillus oryzae bacterial solution is 3×10 9 -6×10 9 CFU / mL; the added amount of the activated Aspergillus oryzae bacterial solution is 2-4% of the mass of the powder; the centrifugation is 7000-9000rpm for 15-20 minutes; the pore size of the microfiltration membrane is 0.22μm; the supernatant is reduced pressure concentrated to 20-25% of the volume; the temperature of the reduced pressure concentration is 45-50℃; the components of the composite filler are (70-80):(20-30) mass ratio of HPD750 macroporous adsorption resin and D101 macroporous adsorption resin.

4. The skin anti-aging and firming composition according to claim 1, wherein The preparation method of the Abelmoschus esculentus seed extract comprises: drying of Abelmoschus esculentus seeds, pulverization into seed powder, mixing of the seed powder with deionized water at a mass ratio of 1: (10-15), adjustment of pH to 4.5-5.0, addition of a composite enzyme, enzymolysis at 50-55℃ for 2-3h; adjustment of temperature to 50-58℃, adjustment of pH to 6.5-7.0, addition of ficin, enzymolysis for 1-1.5h, enzyme inactivation; adjustment of temperature to 50-55℃, adjustment of pH to 7.0-7.5, addition of subtilisin, enzymolysis for 1-1.5h, enzyme inactivation, cooling to room temperature, addition of anhydrous ethanol, stirring extraction, centrifugation, collection of the supernatant, concentration under reduced pressure, loading onto an LH-20 dextran gel column, elution of impurities by 2-3BV of deionized water, further elution by 3-4BV of 50-60% volume concentration ethanol aqueous solution, collection of the eluate, removal of ethanol under reduced pressure, freeze-drying to obtain the extract.

5. The skin anti-aging and firming composition according to claim 4, wherein The particle size of the seed powder is 60-80 mesh; the complex enzyme is cellulase and pectinase in a mass ratio of (1-1.5):1; the adding amount of the complex enzyme is 2-3% of the mass of the seed powder; the adding amount of the ficin is 1-1.5% of the mass of the seed powder; the adding amount of the subtilisin is 1-1.5% of the mass of the seed powder; the enzyme inactivation is all at 85-90℃ for 10-15 minutes; the adding amount of the anhydrous ethanol is 8-10 times of the mass of the seed powder; the stirring extraction is at 200-300 rpm for 1-1.5 hours; the centrifugation is at 4000-5000 rpm for 15-20 minutes; the supernatant is reduced pressure concentrated to 20-25% of the volume; and the temperature of the reduced pressure concentration is all 45-50℃.

6. The skin anti-aging and firming composition according to claim 1, wherein The preparation method of the H. exilis extract comprises the following steps: grinding and homogenizing H. exilis meat to obtain a homogenate, mixing the homogenate with deionized water in a mass ratio of 1:(12-15), adjusting pH to 8.0-8.5, adding trypsin, carrying out enzymolysis at 37-40℃ for 1.5-2 hours, inactivating the enzyme, reducing the temperature to room temperature, adjusting pH to 6.5-7.5, adding an earthworm kinase, carrying out enzymolysis at 37-40℃ for 1.5-2 hours, inactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, ultrafiltering through a 3kDa ultrafilter membrane, taking a component below 3kDa, and freeze-drying to obtain the H. exilis extract.

7. The skin anti-aging and firming composition according to claim 6, wherein The adding amount of the trypsin is 0.8-1.2% of the mass of the homogenate; the adding amount of the earthworm kinase is 0.5-1.0% of the mass of the homogenate; and the enzyme inactivation is all at 85-90℃ for 10-15 minutes.

8. The skin anti-aging and firming composition according to claim 1, wherein The preparation method of the carnosine-zinc gluconate chelate comprises the following steps: dissolving carnosine in 20-25 times of the mass of deionized water in a mass ratio of carnosine: zinc gluconate=(1.5-1.8):1 to obtain a carnosine solution; dissolving zinc gluconate in 15-20 times of the mass of deionized water to obtain a zinc gluconate solution; under stirring, adding the zinc gluconate solution into the carnosine solution, adjusting pH to 5.7-6.3, stirring at 30-35℃ for 1.5-2 hours, reducing pressure to concentrate to obtain a concentrated solution, adding anhydrous ethanol, stirring to crystallize, and after suction filtration, freeze-drying to obtain the carnosine-zinc gluconate chelate.

9. The skin anti-aging and firming composition according to claim 8, wherein The stirring speed is 300-400 rpm; the reducing pressure concentration is at 40-45℃ to 25-30% of the volume; and the adding amount of the anhydrous ethanol is 3-4 times of the volume of the concentrated solution.

10. A method of preparing an anti-aging skin tightening composition according to claim 1, characterized in that, The method comprises the following steps: The fruit axis extract of K. frutescens, the okra seed extract, the tetrapeptide-4, the acetyl octapeptide-1, the H. exilis extract, the paeonol, and the carnosine-zinc gluconate chelate are dispersed or dissolved in a solvent and then mixed to obtain the composition.

Images