Anti-aging and skin-tightening composition and preparation method thereof

Abstract

The invention provides an anti-aging and skin-tightening composition and a preparation method thereof, and belongs to the technical field of anti-aging and skincare, and the composition comprises a hovenia acerba infructescence extract, an okra seed extract, tetrapeptide-4, acetyl octapeptide-1, a hill-pit snail extract, paeonol and a carnosine-zinc gluconate chelate. And carrying out aerobic fermentation on the raisin tree infructescence powder through aspergillus oryzae, and purifying the centrifuged supernatant through a composite filler resin column to obtain the raisin tree infructescence extract. The preparation method comprises the following steps: sequentially carrying out enzymolysis on okra seeds by virtue of compound enzyme, ficin and subtilisin, adding absolute ethyl alcohol for extraction, centrifuging supernate, and purifying by virtue of an LH-20 sephadex column, so as to obtain the okra seed extract. The preparation method comprises the following steps: sequentially carrying out enzymolysis on the mountain snail meat through trypsin and lumbrukinase, centrifuging the enzymatic hydrolysate, and carrying out ultrafiltration on the supernate to obtain the mountain snail extract with the molecular weight of less than 3kDa. The components are matched for use, so that the balance of high-efficiency anti-aging and low-irritation can be realized through synergism of three major pathways of inhibiting collagen degradation, promoting collagen synthesis and resisting sugar and relieving.

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 degrade the structure of the dermis, and finally show 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 tightness. 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 exfoliation 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] 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 Montitriton extract, and uses them in combination with tetrapeptide-4, acetyl octapeptide-1, paeonol, and zinc carnosine-glucose chelate, to achieve synergy through the three pathways of "inhibiting collagen degradation, promoting collagen synthesis, and anti-glycation soothing", breaking through the limitations of traditional single action "anti-aging is not comprehensive"; through "high-purity active ingredients + mild action mechanism", the balance between "high-efficiency anti-aging" and "low irritation" is achieved, and it is suitable for various skin types such as sensitive skin. An anti-aging skin tightening composition, the composition comprising the following raw materials in mass fractions: 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 Montitriton extract, 0.1-0.2 parts of paeonol, and 0.5-1 parts of zinc carnosine-glucose chelate. The preparation of the Haloxylon ammodendron inflorescence axis extract comprises: aerobic fermentation of Haloxylon ammodendron inflorescence axis powder with Aspergillus oryzae, centrifugation of the supernatant, and purification of the supernatant with 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 okra seed extract comprises: sequential enzymolysis of okra seeds with a composite enzyme, bromelin, and subtilisin, addition of anhydrous ethanol for extraction, centrifugation of the supernatant, and purification of the supernatant with an LH-20 dextran gel column to obtain the product; the composite enzyme includes cellulase and pectinase. The preparation of the Montitriton extract comprises: sequential enzymolysis of Montitriton meat with trypsin and earthworm kinase, centrifugation of the supernatant of the enzymolysis solution, and ultrafiltration to obtain a product with a molecular weight of less than 3kDa.

[0007] In the above composition, the preparation method of the Haloxylon ammodendron inflorescence axis extract comprises: drying the Haloxylon ammodendron inflorescence axis, crushing it into powder, mixing the powder with deionized water at a mass ratio of 1: (8-10), sterilizing and cooling, adding tryptone and potassium dihydrogen phosphate, adding Aspergillus oryzae activation bacteria liquid, and performing aerobic fermentation at 28-32℃ for 72-80h to obtain a fermentation liquid; centrifuging, filtering the supernatant with a microfiltration membrane, sterilizing, reducing pressure, concentrating, loading onto a composite filler resin column, washing with 1.5-2BV of 10-15% volume concentration ethanol aqueous solution to remove impurities, then eluting with 3-4BV of 60-65% volume concentration ethanol aqueous solution, collecting the eluate, removing ethanol under reduced pressure, and freeze-drying to obtain the extract.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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℃.

[0012] In the composition, the preparation method of the sinum extract includes the following steps: grinding and homogenizing sinum meat to obtain sinum paste, mixing the sinum paste with deionized water at a mass ratio of 1: (12-15), adjusting the pH to 8.0-8.5, adding trypsin, and enzymolysis 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 eelase, and enzymolysis at 37℃-40℃ for 1.5 h-2 h, inactivating the enzyme, reducing the temperature to room temperature, centrifugation, taking the supernatant, ultrafiltration through a 3 kDa ultrafiltration membrane, taking the component below 3 kDa, and freeze-drying to obtain the sinum extract.

[0013] In the preparation method of the sinum extract, the amount of trypsin added is 0.8%-1.2% of the mass of the sinum paste; the amount of eelase added is 0.5%-1.0% of the mass of the sinum paste; 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.

[0014] 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 obtaining a filter cake after suction filtration, and freeze-drying to obtain the carnosine-zinc gluconate chelate.

[0015] 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.

[0016] The preparation method of the composition for anti-aging and skin tightening includes the following steps: The composition is prepared by dispersing or dissolving, in a solvent, the extract of the fruiting cluster 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.

[0017] The composition is compounded with pharmaceutically or cosmetically acceptable ingredients to prepare a product for anti-aging and skin tightening.

[0018] The composition and the preparation method of the composition for anti-aging and skin tightening provided by the application have the following beneficial effects: Firstly, the composition of the application breaks through the limitation of traditional single action "anti-aging is not comprehensive" through the synergy of the three paths of "inhibiting collagen degradation, promoting collagen synthesis, and anti-glycation soothing". The irritating ingredients such as retinol and high-concentration acid are abandoned, and all the raw materials are obtained from natural biological technology. Through the "high-purity active ingredients + mild action mechanism", the balance between "high-efficiency anti-aging" and "low irritation" is achieved, and the composition is suitable for various skin conditions such as sensitive skin.

[0019] Secondly, the extract of the fruiting cluster of Kalidium foliatum is subjected to aerobic fermentation of Aspergillus oryzae (decomposition of 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 (adsorption of active ingredients with different polarities to remove impurities), and is a strong free radical scavenger (antioxidant) and MMP-1 inhibitor (reduces collagen degradation), which provides the core support for anti-aging.

[0020] Thirdly, the extract of the seed of Abelmoschus esculentus is subjected to cellulase-pectinase (decomposition of cell walls), ficin, and subtilisin (stepwise enzymatic hydrolysis to release effective active peptides) + LH-20 purification (separation of active peptides and small molecular impurities), which assists in enhancing the skin barrier repair ability and synergistically improves the anti-aging stability with other ingredients.

[0021] Fourthly, tetrapeptide-4 and acetyl octapeptide-1 act on fibroblasts to specifically promote collagen synthesis, and have mild and non-irritating effects, which provide a precise target for the "promoting synthesis" path.

[0022] Fifthly, the extract of Facelinidae is subjected to trypsin-ase and lumbrokinase double enzymolysis (sufficient decomposition of proteins into small molecular active peptides) + 3kDa ultrafiltration (removal of macromolecular impurities and retention of active components), which has good anti-aging effect, but no obvious irritation, solves the defects of traditional retinoids such as poor stability and strong irritation, and supplements the mildness of the "promoting synthesis" path.

[0023] Sixthly, zinc carnosine-glucose chelate is chelated in a specific mass ratio (optimizing chelation efficiency and improving stability), which specifically inhibits advanced glycation end products, reduces collagen cross-linking and skin relaxation, and enhances the efficacy of the "anti-glycation soothing" path.

[0024] 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.

[0025] 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

[0026] The present application will be further described below in combination with specific implementation cases, but the present application is not limited to these examples.

[0027] Example 1 An anti-aging and skin tightening composition, comprising the following mass fractions of raw materials: 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.

[0028] The preparation method of the Kalidium foliatum fruit axis extract comprises: Strain activation: Aspergillus oryzae is activated and cultured with PDA liquid medium to obtain Aspergillus oryzae activated bacteria liquid with a bacteria content of 4x10 9 CFU / mL.

[0029] Fermentation extraction: After the Kalidium foliatum fruit axis is dried and crushed through a 50-mesh sieve, powder is obtained, and 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 Aspergillus oryzae activated bacteria liquid are added to the powder, and aerobic fermentation is carried out at 30℃ for 76 h to obtain a fermentation liquid.

[0030] 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 for swelling, and then washed with anhydrous ethanol until the water added to the effluent was not white and turbid, and then washed with deionized water to remove ethanol, and then used); 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 Kalidium foliatum.

[0031] The preparation method of the okra seed extract comprises the following steps: 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.

[0032] 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 the mixture is centrifuged at 4500 rpm for 18 min, and then the supernatant is taken, and then the supernatant is concentrated to 22% of the volume at 48°C under reduced pressure, and then the supernatant is loaded onto an LH-20 dextran gel column, and then the column is eluted with 2.5 BV of deionized water to remove impurities, and then the column is 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 the eluate is freeze-dried to obtain the okra seed extract.

[0033] The preparation method of the mountain snail extract comprises the following steps: 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 lumbriconase 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.

[0034] Purification: the enzymolysis solution is centrifuged at 8200 rpm for 12 min, and then the supernatant is taken, and then the supernatant is ultrafiltered through a 3-kDa ultrafiltration membrane, and then the components below 3 kDa are taken, and then the components are freeze-dried to obtain the mountain snail extract.

[0035] The preparation method of carnosine-zinc gluconate chelate comprises the following steps: Liquid preparation: carnitine is dissolved in 23 times the mass of deionized water according to the mass ratio of carnitine to zinc gluconate of 1.6:1 to obtain a carnitine solution; zinc gluconate is dissolved in 18 times the mass of deionized water to obtain a zinc gluconate solution.

[0036] Chelation: under the condition of stirring at 350 rpm, the zinc gluconate solution is added to the carnitine solution, the pH is adjusted to 6.0, stirring is carried out at 32°C and 350 rpm for 1.5 h, and then 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 the solution is stirred to precipitate crystals; after suction filtration, a filter cake is obtained, which is freeze-dried to obtain the carnosine-zinc gluconate chelate.

[0037] Example 2 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.

[0038] The preparation method of the Haloxylon ammodendron fruit axis extract comprises the following steps: 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.

[0039] Fermentation extraction: after the Haloxylon ammodendron fruit axis is dried, it is crushed to pass through a 60-mesh sieve to obtain a powder; the powder is mixed with deionized water at 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 carried out at 28°C for 72 h to obtain a fermentation liquid.

[0040] Purification: centrifugation is carried out at 7000 rpm for 20 min, the supernatant is filtered through a 0.22-μm microfiltration membrane to remove bacteria; concentration is carried out under reduced pressure at 50°C to 25% of the original volume; loading is carried out on a composite filler resin column (HPD750 macroporous adsorption resin:D101 macroporous adsorption resin=80:20 mass ratio, swelled by soaking in 95% volume concentration ethanol aqueous solution for 24 h, and then washed with anhydrous ethanol until the water added to the effluent does not become white and turbid, and then washed with deionized water to remove ethanol); impurities are removed by elution with 1.5 BV of 15% volume concentration ethanol aqueous solution, and then elution is carried out with 3 BV of 65% volume concentration ethanol aqueous solution; the eluate is collected, ethanol is removed under reduced pressure at 50°C, and freeze-drying is carried out to obtain the Haloxylon ammodendron fruit axis extract.

[0041] The preparation method of the Abelmoschus esculentus seed extract comprises the following steps: Enzymatic hydrolysis: The dried okra seed was crushed and broken into pieces, and then passed through an 80-mesh sieve to obtain seed powder. The seed powder was mixed with deionized water at a mass ratio of 1:10, the pH was adjusted to 5.0, and 2% of a compound enzyme (cellulase: pectinase = 1.5:1) was added to the seed powder. Enzymatic hydrolysis was performed at 50℃ for 3h. The temperature was increased to 50℃, the pH was adjusted to 7.0, and 1% of ficin was added to the seed powder. Enzymatic hydrolysis was performed for 1.5h, and the enzyme was inactivated at 85℃ for 15min. The temperature was decreased to 50℃, the pH was adjusted to 7.0, and 1.5% of bacillus subtilis protease was added to the seed powder. Enzymatic hydrolysis was performed for 1h, and the enzyme was inactivated at 90℃ for 10min. The temperature was decreased to room temperature.

[0042] Extraction and purification: 10 times the mass of anhydrous ethanol was added to the seed powder, and extraction was performed at 200rpm for 1.5h. Centrifugation was performed at 4000rpm for 20min, and the supernatant was collected. Concentration was performed at 50℃ under reduced pressure to 25% of the volume. The sample was loaded onto an LH-20 dextran gel column, and elution was performed with 2BV of deionized water to remove impurities. Elution was then performed with 4BV of 50% volume concentration ethanol aqueous solution. The eluate was collected, and ethanol was removed by concentration at 50℃ under reduced pressure. Freeze-drying was performed to obtain the okra seed extract.

[0043] The preparation method of the sinotaenius gmelini extract includes: Enzymatic hydrolysis: The washed sinotaenius gmelini meat was crushed and homogenized to obtain meat slurry. The meat slurry was mixed with deionized water at a mass ratio of 1:12, the pH was adjusted to 8.0, and 0.8% of trypsin was added to the meat slurry. Enzymatic hydrolysis was performed at 38℃ for 1.5h, and the enzyme was inactivated at 90℃ for 10min. The temperature was decreased to room temperature, the pH was adjusted to 6.5, and 0.5% of an earthworm kinase was added to the meat slurry. Enzymatic hydrolysis was performed at 38℃ for 1.5h, and the enzyme was inactivated at 90℃ for 10min. The enzymatic hydrolysis liquid was obtained.

[0044] Purification: The enzymatic hydrolysis liquid was centrifuged at 8000rpm for 10min, and the supernatant was collected. Ultrafiltration was performed using a 3kDa ultrafiltration membrane, and the components below 3kDa were collected. Freeze-drying was performed to obtain the sinotaenius gmelini extract.

[0045] The preparation method of the carnosine-zinc gluconate chelate includes: Liquid preparation: Carnosine was dissolved in 25 times the mass of deionized water at a mass ratio of carnosine: zinc gluconate = 1.5:1 to obtain a carnosine solution. Zinc gluconate was dissolved in 20 times the mass of deionized water to obtain a zinc gluconate solution.

[0046] Chelation: The zinc gluconate solution was added to the carnosine solution under stirring at 300rpm, the pH was adjusted to 5.7, and stirring was performed at 30℃ and 300rpm for 1.5h. Concentration was performed at 45℃ under reduced pressure to 25% of the volume to obtain a concentrated liquid. Anhydrous ethanol was added to the concentrated liquid at 4 times the volume, and crystallization was performed under stirring. The filter cake was obtained after suction filtration, and freeze-drying was performed to obtain the carnosine-zinc gluconate chelate.

[0047] Example 3 A kind of anti-aging skin tightening composition, including the following mass parts of raw materials: 8 parts of Haloxylon ammodendron axis of inflorescence extract, 3 parts of okra seed extract, 2 parts of tetrapeptide-4, 1 part of acetyl octapeptide-1, 1.5 parts of Rissoa amoenus extract, 0.1 parts of paeonol, 1 parts of carnosine-zinc gluconate chelate.

[0048] The preparation method of the Haloxylon ammodendron axis of inflorescence extract comprises: Strain activation: Aspergillus oryzae is activated and cultured with PDA liquid medium to obtain Aspergillus oryzae activated bacteria liquid with a bacteria content of 6×10 9 CFU / mL.

[0049] Fermentation extraction: after Haloxylon ammodendron axis of inflorescence is dried, it is crushed to pass through a 40-mesh sieve to obtain a powder, the powder is mixed with deionized water at a mass ratio of 1:10, sterilized at 125℃ for 15 min, and cooled to 32℃; 0.5% of tryptone is added to the mass of the powder, 0.1% of potassium dihydrogen phosphate is added to the mass of the powder, and 2% of Aspergillus oryzae activated bacteria liquid is added to the mass of the powder, and aerobic fermentation is carried out at 32℃ for 80 h to obtain a fermentation liquid.

[0050] Purification: centrifugation is carried out at 9000 rpm for 15 min, the supernatant is filtered through a 0.22 μm microfiltration membrane to remove bacteria; 20% of the original volume is concentrated at 45℃ under reduced pressure, and then loaded onto a composite filler resin column (HPD750 macroporous adsorption resin:D101 macroporous adsorption resin=70:30 mass ratio, soaked with 90% volume concentration ethanol aqueous solution for 26 h, swelled, washed with anhydrous ethanol until the water added to the effluent is not white turbid, and then washed with deionized water to remove ethanol), eluted 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 is collected, concentrated to remove ethanol at 45℃ under reduced pressure, and freeze-dried to obtain the Haloxylon ammodendron axis of inflorescence extract.

[0051] The preparation method of the okra seed extract comprises: Enzymatic hydrolysis: after okra seeds are dried, the seeds are crushed and the shells are broken to pass through a 60-mesh sieve to obtain seed powder, the seed powder is mixed with deionized water at a mass ratio of 1:15, the pH is adjusted to 4.5, 3% of a composite enzyme (cellulase: pectinase=1:1) is added to the mass of the seed powder, and enzymatic hydrolysis is carried out at 55℃ for 2 h; the temperature is raised to 58℃, the pH is adjusted to 6.5, 1.5% of ficin is added to the mass of the seed powder, and enzymatic hydrolysis is carried out for 1 h, and the enzyme is inactivated at 90℃ for 10 min; the temperature is lowered to 55℃, the pH is adjusted to 7.5, 1% of subtilisin is added to the mass of the seed powder, and enzymatic hydrolysis is carried out for 1.5 h, the enzyme is inactivated at 85℃ for 15 min, and the temperature is lowered to room temperature.

[0052] Extraction and purification: 8 times the mass of the seed powder of anhydrous ethanol was added, and the mixture was stirred at 300 rpm for 1 h, centrifuged at 5000 rpm for 15 min, and the supernatant was collected. The supernatant was concentrated to 20% of the original volume at 45°C under reduced pressure, and then loaded onto an LH-20 dextran gel column. The column was washed with 3 BV of deionized water to remove impurities, and then eluted with 3 BV of 60% (by volume) ethanol aqueous solution. The eluate was collected, concentrated to remove ethanol at 45°C under reduced pressure, and then freeze-dried to obtain the okra seed extract.

[0053] The preparation method of the Oncomelania hupensis extract includes the following steps: Enzymatic hydrolysis: The washed Oncomelania hupensis meat was ground and homogenized to obtain a slurry. The slurry was mixed with deionized water at a mass ratio of 1:15, and the pH was adjusted to 8.5. Then, 1.2% of the mass of the slurry of trypsin was added, and the mixture was hydrolyzed at 40°C for 2 h. The enzyme was inactivated at 85°C for 15 min, and then the temperature was reduced to room temperature. The pH was adjusted to 7.5, and 1.0% of the mass of the slurry of aesculin was added. The mixture was hydrolyzed at 40°C for 2 h, and the enzyme was inactivated at 85°C for 15 min. The resulting hydrolysate was obtained.

[0054] Purification: The hydrolysate was centrifuged at 8500 rpm for 15 min, and the supernatant was collected. The supernatant was ultrafiltered through a 3 kDa ultrafiltration membrane, and the components below 3 kDa were collected. The collected components were freeze-dried to obtain the Oncomelania hupensis extract.

[0055] The preparation method of the Oncomelania hupensis extract includes the following steps: Liquid preparation: Carbenoxolone was dissolved in 20 times the mass of deionized water to obtain a carbenoxolone solution. Zinc gluconate was dissolved in 15 times the mass of deionized water to obtain a zinc gluconate solution.

[0056] Chelation: The zinc gluconate solution was added to the carbenoxolone solution under stirring at 400 rpm, and the pH was adjusted to 6.3. The mixture was stirred at 35°C for 2 h, and then concentrated to 30% of the original volume at 40°C under reduced pressure to obtain a concentrated solution. Anhydrous ethanol was added to the concentrated solution at a volume of 3 times the original volume, and the mixture was stirred to precipitate crystals. The precipitate was collected by suction filtration to obtain a filter cake, which was freeze-dried to obtain the carbenoxolone-zinc gluconate chelate.

[0057] The preparation method of the anti-aging and skin tightening composition of each of the above embodiments includes the following steps: The fruit axis extract of Kalimeris indica, the okra seed extract, the tetrapeptide-4, the acetyl octapeptide-1, the Oncomelania hupensis extract, the paeonol, and the carbenoxolone-zinc gluconate chelate were dispersed or dissolved in a solvent according to the mass fractions in each embodiment, and then mixed to obtain the composition.

[0058] The anti-aging and skin tightening composition of each of the above embodiments was compounded with ingredients that are pharmaceutically or cosmetically acceptable to prepare a product for anti-aging and skin tightening.

[0059] Comparative Example 1 The extract of Alnus henryi fruiting axis was modified to 9 parts, and the extract of Abelmoschus esculentus seed was modified to 1.5 parts; other parameters and methods were the same as in Example 1.

[0060] Comparative Example 2 The extract of Alnus henryi fruiting axis was modified to 1.2 parts, and the extract of Satsumaia decollata was modified to 6.5 parts; other parameters and methods were the same as in Example 1.

[0061] Comparative Example 3 The extract of Abelmoschus esculentus seed was modified to 1 part, and the extract of Satsumaia decollata was modified to 4.2 parts; other parameters and methods were the same as in Example 1.

[0062] Comparative Example 4 The extract of Satsumaia decollata was modified to 0.5 parts, and the zinc chelate of carnosine-glucose acid was modified to 1.5 parts; other parameters and methods were the same as in Example 1.

[0063] Comparative Example 5 In the preparation of the extract of Alnus henryi fruiting axis, Aspergillus oryzae was replaced by Lactobacillus plantarum (anaerobic fermentation); other parameters and methods were the same as in Example 1.

[0064] Comparative Example 6 In the preparation of the extract of Alnus henryi fruiting axis, the resin column was only filled with HPD750 macroporous adsorption resin; other parameters and methods were the same as in Example 1.

[0065] Comparative Example 7 In the preparation of the extract of Alnus henryi fruiting axis, the resin column was only filled with D101 macroporous adsorption resin; other parameters and methods were the same as in Example 1.

[0066] Comparative Example 8 In the preparation of the extract of Alnus henryi fruiting axis, HPD750 macroporous adsorption resin was replaced by AB-8 macroporous adsorption resin; other parameters and methods were the same as in Example 1.

[0067] Comparative Example 9 In the preparation of the extract of Abelmoschus esculentus seed, ficin was replaced by bromelain; other parameters and methods were the same as in Example 1.

[0068] Comparative Example 10 In the preparation of the extract of Abelmoschus esculentus seed, subtilisin was replaced by papain; other parameters and methods were the same as in Example 1.

[0069] Comparative Example 11 In the preparation of the extract of Abelmoschus esculentus seed, subtilisin was not used for enzymolysis; other parameters and methods were the same as in Example 1.

[0070] Comparative Example 12 In the preparation of the extract of Abelmoschus esculentus seed, LH-20 dextran gel column was replaced by G-25 dextran gel; other parameters and methods were the same as in Example 1.

[0071] Comparative Example 13 In the preparation of the Thais clavigera extract, the earthworm kinase was replaced by bromelain; other parameters and methods were the same as in Example 1.

[0072] Comparative Example 14 In the preparation of the Thais clavigera extract, no trypsin was used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0073] The raw materials involved in the above examples and comparative examples are as follows: Tetrapeptide-4 was from Chengdu Yunxi Chemical Co., Ltd. Acetyl octapeptide-1 was from Chengdu Kecheng Fine Chemical Co., Ltd. Paeonol was from Jiangxi Hairui Natural Plant Co., Ltd. Aspergillus oryzae was from Kolang Biotech (Shanghai) Co., Ltd., product number BNCC 142787. Tryptone was from Jinan Huerui Biotechnology Co., Ltd., source YD-02. Cellulase was from Shandong Yaqiu Biotechnology Co., Ltd., enzyme activity 100,000 U / g. Pectinase was from Wuhan Kairun 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. Earthworm kinase was from Wuhan Xinxinjiali Biotechnology Co., Ltd., enzyme activity 20,000 iu / mg. Papain was from Xi'an Kangpulais 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 Kolang Biotech (Shanghai) Co., Ltd., BNCC 138404.

[0074] I. Cytotoxicity test: 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 to remove bacteria.

[0075] Experimental procedures include: HaCaT (human immortalized keratinocytes) and HDF cells (human dermal fibroblasts) were subjected to proliferation rate test respectively. Cells were cultured in DMEM medium (containing 10% FBS, 1% penicillin-streptomycin) at 37°C, 5% CO2 to logarithmic growth phase. Cells were seeded at a density of 5×10 3 Cells were seeded at a density of 5×10 4 cells / well in 96-well plates, 100 μL of medium per well, and cultured for 24 hours to allow the cells to adhere fully. The old medium was discarded and replaced with fresh medium containing 100 μg / mL of the sample, 100 μL per well. Six replicates were set for each group, and a blank control group (containing only DMEM medium) and a cell base control group (DMEM medium + cells without composition) were also set. The culture was continued at 37°C, 5% CO2 for 24 hours. 10 μL of CCK-8 solution was added to each well, incubated for 2 hours, and the absorbance (OD value) at 450 nm was measured using a microplate reader.

[0076] Evaluation index: cell survival rate (%) = (OD sample group - OD blank control group) / (OD cell base control group - OD blank control group) x 100%.

[0077] II. Collagen synthesis promotion detection: Sample preparation: The composition powder was diluted with DMEM medium to a concentration of 250 μg / mL of sample solution, and sterilized by filtering through a 0.22 μm filter membrane.

[0078] Experimental procedures include: HDF cells (human dermal fibroblasts) were cultured, and when the cell confluence reached more than 85%, they were digested and resuspended. Cells were seeded at a density of 5×10 4 cells / well in 24-well plates, 1 mL of medium per well, and cultured for 24 hours. The old medium was discarded and 1 mL of sample solution was added, with 3 parallel wells set for each concentration. A blank control group (only DMEM medium was added without composition) was also set, and the culture was continued for 48 hours. The cell culture supernatant was collected and the content of type I collagen was detected by ELISA: according to the instructions of the ELISA kit, standard and test culture were added, incubated, washed, and then enzyme-labeled antibody was added. After incubation and washing again, substrate was added for color development, and the absorbance value was measured at 450 nm wavelength. The collagen concentration was calculated according to the standard curve.

[0079] Evaluation index: Collagen synthesis promotion rate (%) = (collagen content of sample group - collagen content of blank group) / collagen content of blank group x 100%.

[0080] III. Free radical scavenging ability detection: Sample preparation: The composition was diluted with absolute ethanol to a concentration of 250 μg / mL of sample solution.

[0081] The experimental procedure includes: 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 in the dark for 30 min. Take anhydrous ethanol as a blank control to adjust zero, and measure the absorbance value at a wavelength of 517 nm: 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.

[0082] Evaluation index: DPPH free radical scavenging rate (%) = [1- (determination group absorbance-sample blank group absorbance) / DPPH blank group absorbance] x 100%.

[0083] Four, matrix metalloproteinase (MMP-1) inhibition detection: 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.

[0084] The experimental procedure includes: 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.

[0085] 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%.

[0086] Five, anti-glycosylation activity detection: 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.

[0087] 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 per tube, gently mixing 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).

[0088] Table 1 Anti-glycosylation reaction system

[0089] 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%.

[0090] Table 2 Test results (average value)

[0091] From the above results, it can be seen that the compositions of Examples 1 to 3 are characterized by: 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 breakage or cytotoxicity caused by excess or deficiency. The fruit stalks of Tribulus terrestris are fermented with Aspergillus oryzae + purified with composite resin, the okra seeds are subjected to three-step enzymatic hydrolysis + LH-20 purification, the mountain snails are subjected to double enzymatic hydrolysis + 3 kDa ultrafiltration, and the carnosine-zinc gluconate is chelated in proportion, and the optimal extraction and purification scheme is designed, resulting in high content of effective active ingredients and few impurities.

[0092] 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 deterioration of the data, and excessive components increase their own irritability and the toxicity of impurities, breaking the balance of "high efficiency-mildness".

[0093] In Comparative Example 5, Aspergillus oryzae is replaced by Lactobacillus plantarum in the preparation of the extract of the fruit stalks of Tribulus terrestris. Aspergillus oryzae is a filamentous fungus that can efficiently decompose the cell wall of the fruit stalks of Tribulus terrestris, releasing active ingredients such as polyphenols, flavonoids, and active peptides, and the fermentation product has no acidic substances that can damage the structure of active ingredients. The main metabolic product of Lactobacillus plantarum is lactic acid, which can lower the pH of the fermentation system, leading to the loss of antioxidant activity of some components and the acquisition of different active peptides; it cannot fully decompose macromolecular substances, resulting in low yield of active ingredients. The antioxidant (DPPH clearance rate), collagen degradation inhibition (MMP-1 inhibition rate), anti-sugar (AGEs inhibition rate), and collagen synthesis promotion (collagen synthesis promotion rate) abilities of the extract are all decreased, and the residual impurities also cause a decrease in cell survival rate.

[0094] The extract of the fruiting branch of Zizyphus jujuba Mill. of Comparative Example 6 and Comparative Example 7 is purified by using only one resin, and the synergistic purification is missing, the types of active ingredients obtained are reduced, and some types of impurities are increased, which affects the overall effect.

[0095] In the purification of the extract of the fruiting branch of Zizyphus jujuba Mill. 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 easily leads to the loss of active ingredients and the stimulation of impurities to cells. All indicators are decreased.

[0096] 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 peptide chains are different, and the efficacy and irritation are different, which leads to changes in data.

[0097] In the purification of the extract of Abelmoschus esculentus 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 extract of Abelmoschus esculentus seed, and effectively separates 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 the adsorption capacity of macromolecular polysaccharides with a molecular weight of >5000 Da is weak, leading to the loss of active ingredients and the residue of impurities. Impurities will interfere with the binding of active ingredients and cell targets, and the absence of macromolecular polysaccharides will weaken the skin barrier repair ability, which together leads to the decrease of all anti-aging indicators; the osmotic pressure of impurities also leads to the decrease of cell survival rate.

[0098] In the preparation of the extract of the mountain snail of Comparative Example 13 and Comparative Example 14, the earthworm kinase 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 peptide chains are different, and the efficacy and irritation are different, which leads to changes in 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 Hemifusus ternatanus extract comprises the following steps: grinding and homogenizing Hemifusus ternatanus meat to obtain a meat slurry, mixing the meat slurry with deionized water in a mass ratio of 1:(12-15), adjusting the pH to 8.0-8.5, adding trypsin, carrying out enzyme hydrolysis at 37-40℃ for 1.5-2 hours, inactivating the enzyme, reducing the temperature to room temperature, adjusting the pH to 6.5-7.5, adding an earthworm kinase, carrying out enzyme hydrolysis at 37-40℃ for 1.5-2 hours, inactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, ultrafiltering the supernatant through a 3kDa ultrafilter membrane, taking a component below 3kDa, and freeze-drying to obtain the Hemifusus ternatanus 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 meat slurry; the adding amount of the earthworm kinase is 0.5-1.0% of the mass of the meat slurry; 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 the 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 fruiting body of Kalimeris indica, the seed extract of Abutilon indicum, tetrapeptide-4, acetyl octapeptide-1, the Hemifusus ternatanus extract, paeonol, and the carnosine-zinc gluconate chelate are dispersed or dissolved in a solvent and then mixed to obtain the composition.

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