An essence with firming and anti-wrinkle effects and a preparation method thereof

Abstract

This invention provides a firming and anti-wrinkle serum and its preparation method, relating to the field of cosmetic technology. The serum comprises: a protein composition, superoxide dismutase (SOD), schistosome extract, budding stalk enzyme polysaccharide, and plant extracts; the protein composition consists of recombinant collagen and fibronectin. The protein composition and SOD form a closed loop of protection and repair, with the former replenishing collagen and filling wrinkles, and the latter scavenging free radicals and protecting collagen from degradation; schistosome extract moisturizes and locks in moisture, strengthening the skin barrier; budding stalk enzyme polysaccharide encapsulates active ingredients, promotes dermal penetration, and provides an immediate firming effect; plant extracts help inhibit collagen degradation and prevent dynamic wrinkles. This serum combines immediate firming and long-term anti-aging repair functions, and can be widely used in firming and anti-wrinkle cosmetics, providing an efficient solution for the field of cosmetic anti-aging.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to an essence with firming and anti-wrinkle effects and its preparation method. Background Technology

[0002] In today's society, with the improvement of people's living standards and the continuous pursuit of beauty, skin care, especially anti-wrinkle and firming, has become a focus of public attention. As the largest organ in the human body, the condition of the skin not only reflects overall health but also directly affects an individual's appearance and self-confidence. However, with age, the skin inevitably shows signs of aging, with wrinkles and sagging being the two main external manifestations of skin aging.

[0003] The skin structure is quite complex, mainly divided into the subcutaneous tissue, dermis, and epidermis. Fibroblasts in the dermis play a crucial role, responsible for synthesizing elastic fibers and collagen. However, between the ages of 20 and 80, the number of fibroblasts decreases significantly, disrupting the balance between the synthesis, maturation, and degradation of elastic fibers and collagen, with a tendency towards degradation. This ultimately leads to loss of dermal elasticity and tension, thinning of the skin, and the formation of wrinkles. With age, the balance between the production and degradation of important substances like collagen in the skin shifts towards degradation, resulting in dermal thinning, tissue damage, skin laxity, and wrinkle formation. Furthermore, ultraviolet radiation, commonly encountered in daily life, also causes significant damage to the skin. Ultraviolet radiation can inhibit collagen synthesis and simultaneously promote the high expression of matrix metalloproteinases, further damaging collagen in the dermis and accelerating the skin aging process.

[0004] Currently available firming and anti-wrinkle cosmetics often fail to meet consumer expectations in actual use. This is primarily due to poor synergy between the ingredients; they not only fail to enhance each other's effectiveness but, in some cases, even interfere with each other, weakening their individual efficacy. Therefore, developing a serum with high-efficiency firming and anti-wrinkle effects that effectively addresses wrinkles and sagging skin, along with its corresponding preparation method, is of paramount importance in the current cosmetics industry. This would not only meet consumer demand for high-quality skincare products but also potentially drive new breakthroughs in the anti-aging field for the entire cosmetics industry. Summary of the Invention

[0005] The purpose of this invention is to provide a serum with firming and anti-wrinkle effects and its preparation method. The serum provided by this invention has excellent firming and anti-wrinkle effects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an essence with firming and anti-wrinkle effects, comprising the following raw materials in parts by weight: 1-3 parts of protein composition, 1-3 parts of superoxide dismutase, 0.5-1 parts of schistosome, 0.3-0.8 parts of budding short stalk enzyme polysaccharide, and 3-6 parts of plant extract; wherein the protein composition is composed of recombinant collagen and fibronectin in a weight ratio of (0.5-1):(0.5-1).

[0008] Preferably, the plant extract is composed of soybean seed extract, chrysanthemum extract and acacia bark extract in a weight ratio of (0.3-0.8):(0.3-0.8):(2-4).

[0009] Preferably, the method for preparing the soybean seed extract includes: extracting soybean seeds with a eutectic solvent solution, purifying them with a macroporous resin, and drying them to obtain the soybean seed extract.

[0010] More preferably, the eutectic solvent consists of choline chloride and ethylene glycol.

[0011] Preferably, the preparation method of the chrysanthemum extract includes: crushing chrysanthemum flowers and extracting them by supercritical CO2 fluid extraction to obtain the chrysanthemum extract.

[0012] Preferably, the preparation method of the Albizia bark extract includes: crushing Albizia bark, mixing it with water, and then subjecting it to anaerobic fermentation by compound microorganisms to obtain the Albizia bark extract.

[0013] More preferably, the composite microorganism consists of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3.

[0014] Preferably, it also includes excipients acceptable in cosmetics.

[0015] The present invention also provides a method for preparing the above-mentioned essence, comprising: mixing and homogenizing a protein composition, superoxide dismutase, schistosome, budding stalk polysaccharide, plant extract and excipients to obtain the essence.

[0016] The present invention also provides the application of the above-mentioned essence in firming and anti-wrinkle cosmetics.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a serum with firming and anti-wrinkle effects, comprising: a protein composition, superoxide dismutase (SOD), schistosome extract, budding stalk enzyme polysaccharide, and plant extracts; the protein composition consists of recombinant collagen and fibronectin. The protein composition and SOD form a closed loop of protection and repair, with the former replenishing collagen and filling wrinkles, and the latter scavenging free radicals and protecting collagen from degradation; schistosome extract moisturizes and locks in moisture, strengthening the skin barrier; budding stalk enzyme polysaccharide encapsulates active ingredients, promotes dermal penetration, and provides an immediate firming effect; plant extracts help inhibit collagen degradation and prevent dynamic wrinkles. In vitro antioxidant tests show that the serum has excellent antioxidant effects and elastase inhibition rate; animal experiments show that the serum of this invention can improve skin elasticity, restore SOD and GSH-Px activity in skin tissue, reduce MDA content, and comprehensively improve the aging state of the skin. This serum of the present invention combines immediate firming and long-term anti-aging repair functions, and can be widely used in firming and anti-wrinkle cosmetics, providing an efficient solution for the field of cosmetic anti-aging. Detailed Implementation

[0019] This invention provides an essence with firming and anti-wrinkle effects, comprising the following raw materials in parts by weight: 1-3 parts of protein composition, 1-3 parts of superoxide dismutase, 0.5-1 parts of schistosome, 0.3-0.8 parts of budding short stalk enzyme polysaccharide, and 3-6 parts of plant extract; wherein the protein composition is composed of recombinant collagen and fibronectin in a weight ratio of (0.5-1):(0.5-1).

[0020] The recombinant collagen in the protein composition of this invention has a structure similar to human collagen, enabling it to effectively penetrate the skin, replenish lost collagen, fill wrinkles, and improve skin elasticity and hydration. Fibronectin, a key protein in the extracellular matrix, promotes skin cell adhesion, migration, and proliferation, guiding and accelerating the more effective repair of collagen and other components. This protein composition directly replenishes skin structural substances, strengthens skin structure, enhances the skin's own resistance, reduces free radicals generated by external stimuli, and indirectly improves the defense efficiency of superoxide dismutase, thus fundamentally improving wrinkles and sagging, providing both immediate and long-term improvement effects.

[0021] The superoxide dismutase described in this invention can efficiently scavenge superoxide free radicals, slow down photoaging and natural aging processes, and provide a stable, low-oxidative-stress microenvironment for the activity and function of the protein composition. This ensures that the supplemented collagen and fibronectin can maintain their biological activity to the maximum extent and effectively integrate into the extracellular matrix of the skin, thereby exerting their best filling and repair effects.

[0022] In this invention, the protein composition and superoxide dismutase have a synergistic effect. Their combination forms a positive cycle and a synergistic closed loop of protection-repair-reprotection. Superoxide dismutase ensures the effective repair work of the protein composition, while the repair work of the protein composition makes the skin healthier and stronger, thereby reducing dependence on external antioxidants. This combination makes the anti-wrinkle effect of the serum not only fast-acting but also provides lasting and fundamental improvement.

[0023] The schistosome extract described in this invention has a strong water-absorbing capacity, forming a breathable moisturizing film on the skin surface to reduce water evaporation. At the same time, it penetrates into the stratum corneum to increase skin hydration. Sufficient moisture can make the skin plump, reduce dryness and fine lines, and activate the proliferation of keratinocytes, enhancing the integrity of the stratum corneum. It also indirectly stimulates the synthesis of dermal mucopolysaccharides, further improving the skin's elasticity and firmness, and forming a dual guarantee of moisture and structural support with collagen.

[0024] The budding short-stalk enzyme polysaccharide described in this invention has excellent biocompatibility and encapsulation properties, enabling it to encapsulate active ingredients such as recombinant collagen and superoxide dismutase within its molecular structure. This prevents the ingredients from being destroyed during storage and use, while simultaneously promoting the slow penetration of active ingredients into the dermis (rather than remaining on the skin surface), thus improving efficacy utilization. On the other hand, the thin film that can be formed on the skin surface has a mild firming effect, instantly improving skin laxity after use. It also gently regulates the skin microenvironment, reducing potential irritation caused by ingredient penetration and avoiding conflicts between anti-wrinkle efficacy and skin tolerance.

[0025] The plant extract of the present invention is preferably composed of soybean seed extract, chrysanthemum extract and acacia bark extract in a weight ratio of (0.3-0.8):(0.3-0.8):(2-4), more preferably 0.5:0.5:3.

[0026] The preferred method for preparing soybean seed extract according to the present invention includes: extracting soybean seeds with a eutectic solvent solution, purifying them with a macroporous resin, and drying them to obtain soybean seed extract; the eutectic solvent is preferably composed of choline chloride and ethylene glycol in a molar ratio of 1:2-4, more preferably 1:3; the water content in the eutectic solvent solution is preferably 10%-20% by mass, more preferably 15%; and the macroporous resin is preferably HPD-600 type macroporous resin.

[0027] This invention enriches soybean isoflavones (phytoestrogens) in soybean seeds through eutectic solvent extraction and macroporous resin purification, promoting the synthesis of collagen and elastin by fibroblasts, while inhibiting the activity of metalloproteinases, reducing collagen loss, and improving skin thickness and elasticity.

[0028] The preferred method for preparing the Globe Amaranth extract of the present invention includes: pulverizing Globe Amaranth flowers and extracting them by supercritical CO2 fluid extraction to obtain the Globe Amaranth extract. More preferably, it includes: pulverizing dried Globe Amaranth flowers through a 40-60 mesh sieve, adding them to a supercritical extraction vessel, and extracting for 2-3 hours at a pressure of 25-35 MPa, a temperature of 35-45°C, and a CO2 flow rate of 20-30 L / h, collecting the Globe Amaranth extract from the bottom of the separation vessel.

[0029] Supercritical CO2 fluid extraction ensures the high bioactivity and high efficiency of the chrysanthemum extract. On the one hand, the chrysanthemum extract of this invention can inhibit the release of inflammatory factors and block the formation of intermediate products of glycation reaction, protecting the collagen structure from damage; on the other hand, it can inhibit the release of neurotransmitters, relax facial expression muscles, and reduce and prevent the formation of dynamic wrinkles.

[0030] The preferred method for preparing the Albizia julibrissin bark extract of the present invention includes: pulverizing Albizia julibrissin bark, mixing it with water, and then subjecting it to anaerobic fermentation by a complex microorganism to obtain the Albizia julibrissin bark extract; the complex microorganism consists of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3; the Lactobacillus rhamnosus LR-K0916 was purchased from the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63006, and is disclosed in patent CN116042470A; the Bifidobacterium bifidum E3 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2022384, and is disclosed in patent CN115261426A.

[0031] The Albizia bark extract obtained through the combined fermentation of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3 utilizes the biotransformation effect of microbial enzymes to improve the dissolution rate of active ingredients. The small molecule components obtained from fermentation decomposition are more likely to penetrate the skin barrier and exert their effects. The transformed components have stronger biological activity, with powerful antioxidant effects, inhibiting the degradation of collagen and elastin, anti-inflammatory and soothing, improving the aging skin environment, improving the skin barrier, and maintaining moisture.

[0032] Among the plant extracts, chamomile extract instantly diminishes existing expression lines by regulating neuromuscular function; soybean seed extract promotes collagen synthesis, increases skin thickness, and improves skin elasticity and firmness; and mimosa bark extract has anti-inflammatory and soothing properties, inhibiting the degradation of elastin and collagen. This invention's plant extracts simultaneously achieve immediate soothing, long-term repair, and comprehensive defense, enabling the serum to not only quickly improve the appearance of wrinkles but also delay the aging process from a physiological perspective, assisting the protein composition and superoxide dismutase in achieving firming and anti-wrinkle effects.

[0033] This invention's serum possesses synergistic anti-aging effects across multiple pathways and levels. It not only significantly lifts the skin and smooths fine lines, but also deeply intervenes in the skin aging process through multi-target physiological mechanisms. By simultaneously supplementing active proteins exogenously and promoting collagen synthesis endogenously, it significantly enhances the structural support of the dermis, achieving improved skin firmness and elasticity. Furthermore, by inhibiting matrix metalloproteinase activity, scavenging free radicals, and blocking the formation of advanced glycation end products, it effectively defends against the degradation of collagen and elastin networks, thereby fundamentally delaying wrinkle formation. In addition, it has excellent moisturizing and barrier repair functions, improving skin hydration and radiance, giving the skin a long-lasting plump and hydrated glow. This invention's serum is a comprehensive anti-aging formulation that combines immediate repair effects with long-term anti-aging repair functions.

[0034] This invention also preferably includes cosmetic-acceptable excipients; the excipients preferably include solvents, humectants, thickeners, pH adjusters, chelating agents, and preservatives; the solvents, humectants, thickeners, pH adjusters, chelating agents, and preservatives are all selected from raw materials known in the cosmetic field. As a preferred embodiment, the solvent includes, but is not limited to, deionized water; the humectant includes, but is not limited to, glycerin, 1,2-butanediol, and panthenol; the thickener includes, but is not limited to, hydroxyethyl cellulose and carbomer; the pH adjuster includes, but is not limited to, sodium hydroxide and citric acid / sodium citrate buffer systems; and the preservative includes, but is not limited to, phenoxyethanol and ethylhexylglycerin.

[0035] The present invention also provides a method for preparing the above-mentioned essence, comprising: mixing and homogenizing a protein composition, superoxide dismutase, schistosome, budding stalk polysaccharide, plant extract and excipients to obtain the essence.

[0036] The present invention also provides the application of the above-mentioned essence in firming and anti-wrinkle cosmetics.

[0037] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0038] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are merely one embodiment of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Recombinant collagen was recombinant human type III collagen, purchased from Shanxi Bohong Biotechnology Co., Ltd.; fibronectin was purchased from Shanxi Bohong Biotechnology Co., Ltd.; superoxide dismutase activity 20,000 IU / g was purchased from Shanxi Bohong Biotechnology Co., Ltd.; *Lactobacillus rhamnosus* LR-K0916 was purchased from Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 63006, published in patent CN116042470A; *Bifidobacterium bifidum* E3 was deposited at China Center for Type Culture Collection, accession number CCTCC NO: M2022384, published in patent CN115261426A; *Lactobacillus plantarum* LP1406 was purchased from China Center for Type Culture Collection, accession number CCTCC NO:M20211500, disclosed in patent CN114891663A; Bifidobacterium longum NX-8 was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCCNo.20116, disclosed in patent CN113832061A.

[0042] The hydroxyl radical scavenging ability test kit, ABTS radical scavenging ability test kit, and DPPH radical scavenging ability test kit were all purchased from Beijing Solarbio Science & Technology Co., Ltd., with catalog numbers BA1255, BC4775, and BC4755, respectively. The rat superoxide dismutase (SOD) ELISA kit, rat glutathione peroxidase (GSH-PX) ELISA kit, and malondialdehyde (MDA) detection kit were all purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., with catalog numbers ml077379, ml107105, and ml094971, respectively.

[0043] Example 1

[0044] Preparation of Firming and Anti-wrinkle Serum

[0045] S1. Preparation of plant extracts

[0046] (1) Preparation of soybean seed extract

[0047] Choline chloride and ethylene glycol were mixed at a molar ratio of 1:3 and stirred at 85°C and 400 rpm for 10 min to obtain a eutectic solvent. The mixture was cooled to 30°C, deionized water was added, and the water mass percentage was adjusted to 15%. The mixture was stirred at 400 rpm for 5 min to obtain a eutectic solvent solution.

[0048] Soybean seeds were crushed and passed through a 200-mesh sieve. A eutectic solvent solution was added at a material-to-liquid ratio of 1:18 g / mL. Extraction was carried out at 60℃ and 400 rpm for 2 hours. The temperature was then lowered to 30℃ and filtered. The filtrate was adsorbed onto an HPD-600 macroporous resin at a flow rate of 2 BV / h. The solution was first eluted with 3 BV of deionized water (2.5 BV / h flow rate) and then with 3 BV of 63% ethanol solution (2 BV / h flow rate). The 63% ethanol eluent was collected and freeze-dried under vacuum to a water content of 2 wt% to obtain soybean seed extract.

[0049] (2) Preparation of Chrysanthemum indicum extract

[0050] The dried chrysanthemum flowers were crushed and passed through a 50-mesh sieve, then added to a supercritical extraction vessel. The extraction was carried out for 2.5 hours at a pressure of 30 MPa, a temperature of 40 °C, and a CO2 flow rate of 25 L / h. The chrysanthemum extract was collected from the bottom of the separation vessel.

[0051] (3) Preparation of Albizia julibrissin bark extract

[0052] The compound microorganism consists of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3. The Lactobacillus rhamnosus LR-K0916 has a bacterial count of 200 million CFU / g and the Bifidobacterium bifidum E3 has a bacterial count of 300 million CFU / g.

[0053] The dried Albizia julibrissin bark was pulverized and passed through a 100-mesh sieve to obtain Albizia julibrissin bark powder. After adding deionized water at a material-to-liquid ratio of 1:20 g / mL, the powder was sterilized. The powder was then inoculated with 5‰ of the weight of the Albizia julibrissin bark powder as a compound microorganism and anaerobic fermented at 37℃ and pH under natural conditions for 60 h. After centrifugation at 8000 rpm for 5 min, the supernatant was sterilized and then freeze-dried under vacuum to a water content of 4 wt% to obtain Albizia julibrissin bark extract.

[0054] (4) Mixing

[0055] Soybean seed extract, chrysanthemum extract and mimosa bark extract were mixed in a weight ratio of 0.5:0.5:3 to obtain plant extracts.

[0056] S2. Weighing

[0057] Recombinant collagen and fibronectin were mixed at a weight ratio of 1:1 to obtain a protein composition;

[0058] Accurately weigh the following per 100 parts by weight: 2 parts protein composition, 2 parts superoxide dismutase, 0.8 parts schistosome, 0.5 parts budding stalk polysaccharide, 5 parts plant extract, 5 parts glycerol, 3 parts 1,2-butanediol, 0.4 parts hydroxyethyl cellulose, 0.8 parts phenoxyethanol, 0.05 parts disodium EDTA, and the balance deionized water.

[0059] S3. Prepare a firming and anti-wrinkle serum

[0060] Glycerin, 1,2-butanediol, and disodium EDTA were added to deionized water and stirred at 500 rpm for 10 min. Hydroxyethyl cellulose was then added and stirred at 1000 rpm for 20 min. Subsequently, protein composition, superoxide dismutase, schistosome, budding stalk polysaccharide, plant extract combination, and phenoxyethanol were added sequentially and stirred at 400 rpm for 30 min. The pH was adjusted to 6.0, and the solution was filtered through a 0.22 μm sterile filter membrane to obtain a firming and anti-wrinkle essence.

[0061] Example 2

[0062] Preparation of Firming and Anti-wrinkle Serum

[0063] S1. Preparation of plant extracts

[0064] (1) Preparation of soybean seed extract

[0065] Choline chloride and ethylene glycol were mixed at a molar ratio of 1:2 and stirred at 80°C and 350 rpm for 13 min to obtain a eutectic solvent. The mixture was cooled to 25°C, deionized water was added, and the water mass percentage was adjusted to 10%. The mixture was stirred at 350 rpm for 8 min to obtain a eutectic solvent solution.

[0066] Soybean seeds were crushed and passed through a 100-mesh sieve. A eutectic solvent solution was added at a material-to-liquid ratio of 1:15 g / mL. Extraction was carried out at 55℃ and 350 rpm for 2.5 h. The temperature was then lowered to 25℃ and filtered. The filtrate was adsorbed onto an HPD-600 macroporous resin at a flow rate of 1.5 BV / h. The solution was first eluted with 2 BV of deionized water (flow rate of 2 BV / h) and then eluted with 2.5 BV of 60% ethanol solution (flow rate of 1.5 BV / h). The 60% ethanol eluent was collected and freeze-dried under vacuum to a water content of 3 wt% to obtain soybean seed extract.

[0067] (2) Preparation of Chrysanthemum indicum extract

[0068] The dried chrysanthemum flowers were crushed and passed through a 40-mesh sieve, then added to a supercritical extraction vessel. The extraction was carried out for 3 hours at a pressure of 25 MPa, a temperature of 35 °C, and a CO2 flow rate of 20 L / h. The chrysanthemum extract was collected from the bottom of the separation vessel.

[0069] (3) Preparation of Albizia julibrissin bark extract

[0070] The compound microorganism consists of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3. The Lactobacillus rhamnosus LR-K0916 has a bacterial count of 150 million CFU / g and the Bifidobacterium bifidum E3 has a bacterial count of 250 million CFU / g.

[0071] The dried Albizia julibrissin bark was pulverized and passed through an 80-mesh sieve to obtain Albizia julibrissin bark powder. After adding deionized water at a material-to-liquid ratio of 1:18 g / mL, the powder was sterilized. The powder was then inoculated with a compound microorganism at 7‰ of the weight of the Albizia julibrissin bark powder and anaerobic fermented at 35℃ and pH under natural conditions for 72 hours. After centrifugation at 7000 rpm for 7 minutes, the supernatant was sterilized and then freeze-dried under vacuum until the water content was 2wt% to obtain Albizia julibrissin bark extract.

[0072] (4) Mixing

[0073] Soybean seed extract, chrysanthemum extract and mimosa bark extract were mixed in a weight ratio of 0.3:0.3:2 to obtain plant extracts.

[0074] S2. Weighing

[0075] Recombinant collagen and fibronectin were mixed at a weight ratio of 0.5:1 to obtain a protein composition.

[0076] Accurately weigh the following per 100 parts by weight: 3 parts protein composition, 1 part superoxide dismutase, 1 part schistosome, 0.3 parts budding stalk polysaccharide, 3 parts plant extract, 7 parts glycerol, 2 parts 1,2-butanediol, 0.7 parts hydroxyethyl cellulose, 0.3 parts phenoxyethanol, 0.07 parts disodium EDTA, and the balance deionized water.

[0077] S3. Prepare a firming and anti-wrinkle serum

[0078] Glycerin, 1,2-butanediol, and disodium EDTA were added to deionized water and stirred at 400 rpm for 15 min. Hydroxyethyl cellulose was then added and stirred at 800 rpm for 30 min. Subsequently, a protein composition, superoxide dismutase, schistosome, budding stalk polysaccharide, a combination of plant extracts, and phenoxyethanol were added and stirred at 300 rpm for 30 min. The pH was adjusted to 5.8, and the solution was filtered through a 0.22 μm sterile filter membrane to obtain a firming and anti-wrinkle essence.

[0079] Example 3

[0080] Preparation of Firming and Anti-wrinkle Serum

[0081] S1. Preparation of plant extracts

[0082] (1) Preparation of soybean seed extract

[0083] Choline chloride and ethylene glycol were mixed at a molar ratio of 1:4 and stirred at 88°C and 450 rpm for 8 min to obtain a eutectic solvent. The mixture was cooled to 32°C, deionized water was added, and the water mass percentage was adjusted to 20%. The mixture was stirred at 450 rpm for 3 min to obtain a eutectic solvent solution.

[0084] Soybean seeds were crushed and passed through a 300-mesh sieve. A eutectic solvent solution was added at a material-to-liquid ratio of 1:20 g / mL. Extraction was carried out at 62℃ and 450 rpm for 1.5 h. The temperature was then lowered to 32℃ and filtered. The filtrate was adsorbed onto an HPD-600 macroporous resin at a flow rate of 2.5 BV / h. The solution was first eluted with 3.5 BV of deionized water (flow rate of 3 BV / h) and then eluted with 3.5 BV of 65% ethanol solution (flow rate of 2.5 BV / h). The 65% ethanol eluent was collected and freeze-dried under vacuum to a water content of 3 wt% to obtain soybean seed extract.

[0085] (2) Preparation of Chrysanthemum indicum extract

[0086] The dried chrysanthemum flowers were crushed and passed through a 60-mesh sieve, then added to a supercritical extraction vessel. Extraction was carried out for 2 hours at a pressure of 35 MPa, a temperature of 45 °C, and a CO2 flow rate of 30 L / h. The chrysanthemum extract was collected from the bottom of the separation vessel.

[0087] (3) Preparation of Albizia julibrissin bark extract

[0088] The compound microorganism consists of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3. The Lactobacillus rhamnosus LR-K0916 has a bacterial count of 300 million CFU / g and the Bifidobacterium bifidum E3 has a bacterial count of 400 million CFU / g.

[0089] The dried Albizia julibrissin bark was pulverized and passed through a 120-mesh sieve to obtain Albizia julibrissin bark powder. After adding deionized water at a material-to-liquid ratio of 1:23 g / mL, the powder was sterilized. The powder was then inoculated with a compound microorganism at 3‰ of the weight of the Albizia julibrissin bark powder and anaerobic fermented at 39℃ and pH under natural conditions for 56 hours. After centrifugation at 9000 rpm for 3 minutes, the supernatant was sterilized and then freeze-dried under vacuum to a water content of 5wt% to obtain Albizia julibrissin bark extract.

[0090] (4) Mixing

[0091] Soybean seed extract, chrysanthemum extract and mimosa bark extract were mixed in a weight ratio of 0.8:0.8:4 to obtain plant extracts.

[0092] S2. Weighing

[0093] Recombinant collagen and fibronectin were mixed at a weight ratio of 1:0.5 to obtain a protein composition;

[0094] Accurately weigh the following per 100 parts by weight: 1 part protein composition, 3 parts superoxide dismutase, 0.5 parts schistosome, 0.8 parts budding stalk polysaccharide, 6 parts plant extract, 3 parts glycerol, 3 parts 1,2-butanediol, 0.3 parts hydroxyethyl cellulose, 0.8 parts phenoxyethanol, 0.03 parts disodium EDTA, and the balance deionized water.

[0095] S3. Prepare a firming and anti-wrinkle serum

[0096] Glycerin, 1,2-butanediol, and disodium EDTA were added to deionized water and stirred at 600 rpm for 8 min. Hydroxyethyl cellulose was then added and stirred at 1100 rpm for 18 min. Subsequently, a protein composition, superoxide dismutase, schistosome, budding stalk polysaccharide, a combination of plant extracts, and phenoxyethanol were added and stirred at 500 rpm for 28 min. The pH was adjusted to 6.2, and the solution was filtered through a 0.22 μm sterile filter membrane to obtain a firming and anti-wrinkle essence.

[0097] Comparative Example 1

[0098] The specific implementation method is the same as in Example 1, except that "2 parts of protein composition and 2 parts of superoxide dismutase" are replaced with "4 parts of protein composition".

[0099] Comparative Example 2

[0100] The specific implementation method is the same as in Example 1, except that "2 parts of protein composition and 2 parts of superoxide dismutase" are replaced with "4 parts of superoxide dismutase".

[0101] Comparative Example 3

[0102] The specific implementation method is the same as that in Example 1, except that the eutectic solvent solution in step (1) of preparing soybean seed extract in S1 is replaced with a 63% ethanol solution by volume.

[0103] Comparative Example 4

[0104] The specific implementation method is the same as in Example 1, except that the preparation method of the chrysanthemum extract in step (2) of S1 is as follows: the dried chrysanthemum flowers are crushed and passed through a 50-mesh sieve, and 75% ethanol solution is added at a material-to-liquid ratio of 1:10 g / mL. The mixture is ultrasonically treated at 45℃, 30kHz, and 200W for 1.5h, centrifuged at 3000r / min for 20min, and the supernatant is freeze-dried under vacuum until the water content is 4wt% to obtain the chrysanthemum extract.

[0105] Comparative Example 5

[0106] The specific implementation method is the same as in Example 1, except that the composite microorganisms in the preparation of Albizia bark extract in step (3) of S1 are composed of Lactobacillus plantarum LP1406 and Bifidobacterium longum NX-8. The number of Lactobacillus plantarum LP1406 in the composite microorganisms is 200 million / g and the number of Bifidobacterium longum NX-8 is 300 million / g. All other parameters are the same as in Example 1.

[0107] Comparative Example 6

[0108] The specific implementation method is the same as in Example 1, except that "0.8 parts of schizocarpine and 0.5 parts of budding short stalk enzyme polysaccharide" is adjusted to "0.2 parts of schizocarpine and 1.1 parts of budding short stalk enzyme polysaccharide".

[0109] Comparative Example 7

[0110] The specific implementation method is the same as in Example 1, except that "0.8 parts of schizotypalin and 0.5 parts of budding short stalk enzyme polysaccharide" is adjusted to "1.2 parts of schizotypalin and 0.1 parts of budding short stalk enzyme polysaccharide".

[0111] Experimental Example 1

[0112] in vitro

[0113] (1) Antioxidant test

[0114] The essences of Examples 1-3 and Comparative Examples 1-7 were mixed with water at a volume ratio of 1:1 and shaken at 2000 rpm for 2 min to obtain the test samples.

[0115] The hydroxyl radical scavenging ability, ABTS radical scavenging ability, and DPPH radical scavenging ability of the test samples in Examples 1-3 and Comparative Examples 1-7 were tested according to the kit instructions. The specific results are shown in Table 1.

[0116] Table 1. Antioxidant capacity of each sample tested

[0117] Table 1 shows that Examples 1-3 exhibit stronger antioxidant capacity than Comparative Examples 1-7. The comparison of data between Comparative Examples 1-2 and Example 1 demonstrates a synergistic effect between the protein composition and superoxide dismutase; the antioxidant effect of either component alone cannot compare to the combination of the two. The comparison of data between Comparative Examples 3-5 and Example 1 indicates that specific extraction methods, specific solvents, and specific complex microorganisms directly affect the antioxidant activity of the extract. The comparison of data between Comparative Examples 6-7 and Example 1 shows that the weight ratio of schistosome extract and budding stalk polysaccharide affects the maintenance of the extract's antioxidant activity. The formulation and preparation process of the extract of this invention collectively endow it with superior in vitro antioxidant capacity, allowing for further in vivo experiments.

[0118] (2) Determination of elastase inhibition rate

[0119] The anti-aging compositions of Examples 1-3 and Comparative Examples 1-7 were mixed with deionized water at a volume ratio of 1:3 to obtain the test samples.

[0120] Add 90 μL of Tris-HCl buffer solution to each well of a 96-plate, followed by 30 μL of the test sample and 10 μL of substrate (nsucc-(ALa)-3-pnitroanilide solution). Mix well and incubate at 25°C for 20 min. Then add 20 μL of porcine pancreatic elastase solution and incubate at 25°C for 10 min. Measure the absorbance at 410 nm. Use deionized water as a blank control instead of the sample.

[0121] Elastase inhibition rate (%) = (1 - absorbance of test sample / absorbance of blank control group) × 100%.

[0122] The results of elastase inhibition rates of the test samples in Examples 1-3 and Comparative Examples 1-7 are shown in Table 2.

[0123] Table 2. Elastase inhibition rate (%) of the tested samples

[0124] Table 2 shows that Examples 1-3 exhibited stronger elastase inhibition rates compared to Comparative Examples 1-7. The comparison of data from Comparative Examples 1-2 with Example 1 indicates that the combination of the protein composition and superoxide dismutase significantly improves the elastase inhibition rate. The comparison of data from Comparative Examples 3-5 with Example 1 demonstrates that specific extraction methods, specific solvents, and specific complex microorganisms directly affect the elastase inhibition rate of the extract. The comparison of data from Comparative Examples 6-7 with Example 1 shows that the weight ratio of schistosome extract to budding stalk polysaccharide affects the elastase inhibition rate of the extract. The extract of this invention exhibits a strong inhibitory effect on elastase and can be further validated through in vivo experiments.

[0125] Experimental Example 2

[0126] animal experiments

[0127] One hundred female SPF-grade SD rats, weighing 220-250g, aged 6 months, were purchased from a qualified laboratory animal center. The enclosure was maintained at a temperature of 22-25℃ and humidity of 45-55%, with a 12-hour light / 12-hour dark cycle. Rats had free access to food and water and were allowed to acclimatize for one week.

[0128] Rats were divided into 10 groups (blank group, model group, Example 1 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Comparative Example 5 group, Comparative Example 6 group and Comparative Example 7 group), with 10 rats in each group.

[0129] Except for the control group, rats in other groups were used to establish a skin aging model by subcutaneous injection of D-galactose into the neck and back, once daily at a dose of 100 mg / kg, for 42 consecutive days. The control group rats were injected subcutaneously into the neck and back with an equal volume of physiological saline during the same period.

[0130] Starting from day 22 of modeling, skin samples were applied to rats in each group. Hair was removed from both sides of the spine on the back of the rats, with a hair removal area of ​​approximately 4cm × 4cm. After cleaning the hair removal area with physiological saline, the corresponding serum was applied to each group at a dosage of 0.2mL / cm². 2 Apply once daily for 21 consecutive days. The control group and model group were treated with an equal volume of deionized water, using the same application method as the other groups.

[0131] (1) Observation and scoring of skin wrinkles

[0132] Two hours after the last application, images of the hair-removed areas on the backs of rats in each group were taken using a skin wrinkle detector. The number, depth, and width of skin wrinkles were observed and scored. The specific results are shown in Table 3.

[0133] The skin wrinkle scoring criteria were as follows: no wrinkles (0 points); very shallow and few wrinkles (1 point); shallow and few wrinkles (2 points); deep and many wrinkles (3 points); deep and many wrinkles (4 points); and very deep and many wrinkles (5 points). The average score of 5 rats in each group was taken as the wrinkle score of that group.

[0134] Table 3 Skin wrinkle scores of rats in each group

[0135] Table 3 shows that the skin wrinkle score of the model group was significantly higher than that of the control group, indicating that D-galactose successfully induced a rat model of skin aging. The score in Example 1 was very close to that of the control group and much lower than that of the model group, indicating that the essence of this invention can extremely effectively improve or even prevent wrinkles caused by aging. Data from Comparative Examples 1-2 and Example 1 show that the protein composition combined with superoxide dismutase has a significant anti-wrinkle effect. Data from Comparative Examples 3-5 and Example 1 show that using a suitable extraction method can improve the anti-wrinkle effect. Data from Comparative Examples 6-7 and Example 1 show that the weight ratio of schistosome extract and budding stalk polysaccharide affects the anti-wrinkle effect of the essence. From the rat experiments, the essence of this invention has a visible and excellent anti-wrinkle effect, and can significantly improve skin wrinkles caused by aging.

[0136] (2) Skin elasticity test

[0137] After taking images of the hair-removed areas on the backs of rats in each group, three test points were randomly selected on the hair-removed areas on the backs of rats in each group using a skin elastometer to measure the skin elasticity parameter R2 value (total skin elasticity). Each test point was measured three times, and the average value was taken as the skin elasticity value of the rat. The average skin elasticity value of 10 rats in each group was then calculated, and the skin elasticity improvement rate of each group of rats was calculated.

[0138] Skin elasticity improvement rate (%) = (total skin elasticity value of the test group - total skin elasticity value of the model group) / total skin elasticity value of the model group × 100%.

[0139] The improvement rate of skin elasticity is shown in Table 4.

[0140] Table 4. Improvement rate of skin elasticity in rats of each group (%)

[0141] Table 4 shows that the serums of Example 1 and Comparative Examples 1-7 all improved skin elasticity, with Example 1 showing the best improvement. A comparison of the data from Comparative Examples 1-2 and Example 1 indicates that supplementing only structural proteins (protein composition) without providing antioxidant protection (superoxide dismutase), or only providing antioxidant protection without supplementing structural proteins, cannot effectively restore skin elasticity, indicating that the protein composition and superoxide dismutase have a synergistic effect in improving skin elasticity. A comparison of the data from Comparative Examples 3-7 and Example 1 shows that the extraction method of the plant extracts and the ratio of auxiliary ingredients play an important role in supporting the dermal structure and restoring elasticity in the serum. The serum of this invention can fundamentally improve the mechanical properties of the skin, achieving a significant firming effect.

[0142] (3) Biochemical index detection

[0143] After the skin elasticity test was completed, blood was collected from the orbital venous plexus, centrifuged at 3000 rpm for 10 minutes, and serum was obtained.

[0144] According to the kit instructions, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) in rat serum were measured. Each indicator was measured three times and the average value was taken. The specific results are shown in Table 5.

[0145] Table 5. Detection of biochemical indicators in the serum of rats in each group

[0146] Table 5 shows that, compared with the control group, the activities of SOD and GSH-Px in the model group decreased sharply, while the content of MDA increased sharply, indicating that D-galactose successfully induced a rat model of skin aging. In Example 1, the activities of SOD and GSH-Px recovered to near normal levels, and the content of MDA also decreased to the normal range. This indicates that the essence of this invention not only provides exogenous antioxidants (such as superoxide dismutase) but also activates or protects the body's own endogenous antioxidant system, thereby comprehensively resisting oxidative stress and protecting skin cells and extracellular matrix (such as collagen and elastin) from damage. A comparison of the data from Comparative Examples 1-2 and Example 1 shows that the protein composition and superoxide dismutase have a synergistic effect, which can alleviate the decrease in SOD and GSH-Px activities and the increase in MDA content caused by skin aging. A comparison of the data from Comparative Examples 3-7 and Example 1 shows that a suitable extraction method for plant extracts and a suitable ratio of auxiliary ingredients have a positive effect on alleviating the decrease in SOD and GSH-Px activities and the increase in MDA content caused by skin aging.

[0147] The above experimental data fully demonstrate that the essence of this invention has excellent firming, anti-wrinkle, and antioxidant effects. The key to its success lies in the synergistic effect between the protein composition and superoxide dismutase. Any alteration to the core ingredients, formulation, or preparation process will lead to a significant decrease in product performance. Specifically, the combination of the protein composition and superoxide dismutase, the combination of three plant extracts prepared using specific processes, and the appropriate addition of schistosome extract and budding stalk polysaccharide are the core components of its high efficiency.

[0148] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An essence having a firming and anti-wrinkle effect, characterized in that, The raw materials include the following components by weight: 1-3 parts of a protein composition, 1-3 parts of superoxide dismutase, 0.5-1 part of lysozyme, 0.3-0.8 part of a dextranase polysaccharide, and 3-6 parts of a plant extract; The protein composition is composed of recombinant human type III collagen and fibronectin at a weight ratio of (0.5-1):(0.5-1); The plant extract is composed of soybean seed extract, spilanthes extract, and albizia bark extract at a weight ratio of (0.3-0.8):(0.3-0.8):(2-4); The preparation method of the soybean seed extract comprises the following steps: extracting soybean seeds from a deep eutectic solvent solution, purifying the extracted soybean seeds by using a macroporous resin, and drying the purified soybean seeds to obtain the soybean seed extract; the deep eutectic solvent is composed of choline chloride and ethylene glycol at a molar ratio of 1:2-4; the mass percentage of water in the deep eutectic solvent solution is 10%-20%; and the macroporous resin is HPD-600 type macroporous resin; The preparation method of the spilanthes extract comprises the following steps: crushing dried spilanthes flowers to pass through a 40-60 mesh sieve, adding the crushed spilanthes flowers into a supercritical extraction kettle, and extracting the spilanthes flowers at a pressure of 25-35 MPa, a temperature of 35-45 ℃, and a CO2 flow rate of 20-30 L / h for 2-3 h to obtain the spilanthes extract from the bottom of the separation kettle; The preparation method of the albizia bark extract comprises the following steps: crushing albizia bark, mixing the crushed albizia bark with water, and subjecting the mixture to anaerobic fermentation by using a composite microorganism to obtain the albizia bark extract; the composite microorganism is composed of Lactobacillus rhamnosus LR-K0916 and Bifidobacterium bifidum E3.

2. The serum of claim 1, wherein, The cosmetic acceptable adjuvant is also included.

3. A method of preparing the serum according to claim 1 or 2, characterized in that, The preparation method comprises the following steps: The protein composition, the superoxide dismutase, the lysozyme, the dextranase polysaccharide, the plant extract, and the adjuvant are mixed and homogenized to obtain the serum.

4. The serum according to claim 1 or 2 in a firming and anti-wrinkle cosmetic.

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