CN111671691B

CN111671691B - Collagenase inhibitor composition, anti-aging face cream and preparation method thereof

Info

Publication number: CN111671691B
Application number: CN202010671184.XA
Authority: CN
Inventors: 谢佩佩; 杨登亮; 李传茂; 张楚标; 曾伟丹; 张伟杰
Original assignee: Guangdong Danz Group Co Ltd; Guangzhou Keneng Cosmetic Research Co Ltd
Current assignee: Guangdong Danz Group Co Ltd; Guangzhou Keneng Cosmetic Research Co Ltd
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2023-12-15
Anticipated expiration: 2040-07-13
Also published as: CN111671691A

Abstract

The application discloses a collagenase inhibitor composition, an anti-aging facial cream and a preparation method thereof, wherein the collagenase inhibitor composition comprises, by mass, 50-99% of licorice root extract, 1-50% of chamomile extract, and the mass ratio of the chamomile extract to the licorice root extract is preferably 1:1 to 9. The anti-aging face cream comprises a collagenase inhibitor composition and a penetration enhancer, wherein the adding amount of the collagenase inhibitor composition is 0.01-10% and the adding amount of the penetration enhancer is 0.01-10% based on the total mass of the anti-aging face cream, the collagenase inhibitor composition comprises 50-99% of licorice root extract, 1-50% of chamomile extract and the penetration enhancer is bis-diethoxydiglycol cyclohexane 1, 4-dicarboxylic acid ester. The collagenase inhibitor composition utilizes the combination of licorice root extract and chamomile extract, has a synergistic effect, and can well inhibit collagenase activity. The anti-aging face cream can reduce the degradation of collagen, has the anti-aging effect, has the functions of moisturizing and locking water, can prevent water loss, and creates a moisturizing barrier for skin.

Description

Collagenase inhibitor composition, anti-aging face cream and preparation method thereof

Technical Field

The application relates to the field of daily chemicals, in particular to a collagenase inhibitor composition, an anti-aging facial cream and a preparation method thereof.

Background

Collagen is the oldest and most abundant extracellular matrix protein, and has wide application in the industries of foods, cosmetics, pharmacy, biomedicine and the like. The collagen family of extracellular matrix proteins plays a vital role in the evolution of multicellular animals. Currently, 28 triple-helical proteins are named collagen and can be divided into several subgroups based on their structural and functional properties. Collagen is mainly produced by fibroblasts of the dermis layer, is a main component of the dermis layer, can maintain the structure of the skin, and imparts toughness and elasticity to the skin. The collagen content and distribution determine whether the skin is youthful. However, abnormal decrease of collagen content causes thinning of dermis, slackening of skin, loss of elasticity, occurrence of wrinkles, and influence of life quality of people. With the continued intensive research on collagen, researchers have found that collagenase plays an important role in the dynamic balance of skin collagen, and that its overexpression and abnormal activation are one of the main causes of skin aging. Therefore, inhibiting collagenase activity can block skin collagen degradation, increase collagen content, and realize anti-aging effect.

Conventionally, in order to prevent skin from sagging, losing elasticity, causing wrinkles, and the like, and to keep skin youthful, anti-aging creams have been proposed which contain various components such as hydrolyzed collagen, hyaluronic acid, polypeptides, retinol, and derivatives thereof. However, if these ingredients are used in large amounts, there are problems in terms of anti-aging effects, skin feel in use, and safety. If the molecular weight of the hydrolyzed collagen is too large, the hydrolyzed collagen is not easy to penetrate through the skin barrier of the human body to reach the dermis layer; hyaluronic acid cannot substantially slow down the loss of collagen; polypeptides and retinol present a certain irritation and safety risk to the skin, etc.

Along with the increase of the attention of people to skin health, the development of the natural anti-aging agent with safety, stability, obvious effect and high cost performance becomes one of the main research directions of the current medicine and cosmetic industry, and has very good development prospect.

Disclosure of Invention

In view of the prior art, the hydrolyzed collagen has excessive molecular weight, and is not easy to penetrate through the skin barrier of the human body to reach the dermis; hyaluronic acid cannot substantially slow down the loss of collagen; the application firstly provides a collagenase inhibitor composition for inhibiting collagenase activity, which has certain problems of irritation and safety risk to skin.

The application also provides the anti-aging facial cream which can slow down the degradation of collagen, has excellent anti-aging effect and has no side effect on human bodies.

The application also provides the anti-aging face cream which is simple to operate and easy to obtain raw materials and the preparation method thereof.

As a first aspect of the present application, the technical solution adopted by the present application to solve the technical problems is: a collagenase inhibitor composition is provided, which comprises licorice root extract and chamomile extract, wherein the licorice root extract accounts for 50-99% and the chamomile extract accounts for 1-50% by mass.

Glycyrrhrizae radix (Glycyrrhiza uralensis Fisch) is perennial herb of Leguminosae and Glycyrrhiza, has strong root and root-like stem, and is a tonic Chinese herbal medicine. The main active ingredient of the licorice root is glycyrrhizin, which is a triterpenoid saponin, and the licorice root can be extracted to 4-10%, and researches show that the glycyrrhizin has glucocorticoid pharmacological effects, and anti-inflammatory, antiviral, antitumor and liver protecting activities. Glycyrrhizin also has important chemopreventive effects on edema removal, oxidation stress induced by glycyrrhizic acid on TPA (12-O-tetradecyl phenol-13-acetate) and skin hyperproliferation markers. In addition, licorice root extract is also effective in treating dermatitis, eczema and psoriasis as well as corticosteroids. Since glycyrrhetinic acid is also an active ingredient of a glycyrrhiza extract, the chemical structure of glycyrrhetinic acid is similar to that of hydrocortisone, and 2% of glycyrrhetinic acid can synergistically increase and resist inflammation of hydrocortisone, so that the glycyrrhetinic acid can be widely applied to the treatment of skin diseases. Glycyrrhizin and glycyrrhetinic acid may be promising candidates for the prevention and treatment of local inflammatory lesions and skin lesions.

The inventors of the present application found that licorice root extract has excellent inhibitory effect on collagenase activity. The collagenase inhibition rate gradually increases with the increase in mass concentration of the licorice root extract.

Chamomile (Matricaria recutita), a plant of the family Compositae, is enriched in sesquiterpenes (e.g. bisphenol, farnesene), sesquiterpenes (e.g. Gan Juxi, matrine), flavonoids (e.g. apigenin, luteolin) and volatile oils. Flos Matricariae Chamomillae is widely used in food, flavoring, spice and cosmetic fields. The chamomile oil vegetable oil can play a role in protecting the skin through a sealing effect, so that the skin keeps moisture and the TEWL value is reduced. Among them, german chamomile oil has an anti-inflammatory ability by alleviating the immunomodulatory potential of atopic dermatitis by affecting Th2 cell activation.

The licorice root extract and the chamomile extract are matched, so that the synergistic effect is achieved, and the collagenase activity can be well inhibited.

Further, the mass ratio of the chamomile extract to the licorice root extract is 1:1 to 99, more preferably 1:1 to 9, still more preferably 1:1 or 1:9.

further, the preparation method of the licorice root extract comprises the following steps:

step one: cleaning Glycyrrhrizae radix, oven drying, and pulverizing;

step two: adding the crushed licorice root raw material into an extraction tank, and extracting by using water and alcohol, wherein the mass ratio of the licorice root raw material to the water to the alcohol is 1:0.1 to 1:0.5 to 2.5, extracting for 2 to 4 times at the extracting temperature of 30 to 60 ℃ for 2 to 8 hours to obtain licorice root extracting solution;

step three: filtering the obtained licorice root extract, and concentrating the filtrate in an external circulation tank and a sedimentation tank to obtain liquid components and precipitate;

and step four, spray drying the liquid component, and drying the precipitate and sieving the precipitate with a 80-100-mesh sieve to obtain the licorice root extract.

In the application, the mass ratio of the licorice root raw material to the water to the alcohol is 1:0.1-1:0.5-2.5; more preferably 1:0.2 to 1: 0.5-1 when the ratio of the liquorice raw material to the water to the alcohol is 1:0.25 to 0.55: when the amount is in the range of 0.5 to 1, the obtained licorice root extract has a high active ingredient, and can further effectively inhibit collagenase activity.

Further, filtering the obtained licorice root extract, storing in a storage tank, allowing filtrate in the storage tank to slowly flow into an external circulation tank and a sedimentation tank for concentration to obtain a liquid component and a precipitate, spray-drying the liquid component, drying the precipitate, and sieving the precipitate with a 80-100-mesh sieve to obtain the licorice root extract with proper particle size.

In the present application, the extraction temperature is 30 to 60 ℃, preferably 40 to 50 ℃. The number of the extraction is 2 to 4, preferably 3 to 4. The extraction time is 2 to 8 hours, preferably 2 to 4 hours. In general, the alcohol may be ethanol, ethylene glycol, propanol, or the like.

The licorice root extract obtained by the preparation method is brown powder, and has the ash content of below 10%, the water content of below 5%, the heavy metal content of below 10ppm, the lead content of below 2ppm, the arsenic content of below 3ppm and the total bacterial count of below 1000 CFU/g; the total number of mould and yeast is below 100CFU/g, and bacteria such as salmonella and escherichia coli are avoided.

As a second aspect of the present application, there is provided an anti-aging cream to which the above-mentioned collagenase inhibitor composition is applied, the anti-aging cream comprising a collagenase inhibitor composition and a penetration enhancer, the collagenase inhibitor composition being added in an amount of 0.01 to 10% and the penetration enhancer being added in an amount of 0.01 to 10% based on the total mass of the anti-aging cream; the collagenase inhibitor composition comprises a licorice root extract and a chamomile extract, wherein the licorice root extract accounts for 50-99% by mass, and the chamomile extract accounts for 1-50% by mass; the penetration enhancer is bis-diethoxydiglycol cyclohexane 1, 4-dicarboxylate.

The collagenase inhibitor composition is added in an amount of 0.01 to 10% based on the total mass of the anti-aging cream, for example: the collagenase inhibitor composition may be added in an amount of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 9%, etc. When the collagenase inhibitor composition is added in an amount of 0.01 to 10%, the skin elasticity of the anti-aging cream is increased. When the addition amount of the collagenase inhibitor composition is less than 0.01%, the skin elasticity improving effect is not obvious, and the anti-aging effect cannot be achieved; when the collagenase inhibitor composition is added in an amount of more than 10%, the content of the composition is too high, the cost is too high, and the anti-aging effect cannot be further increased.

The penetration enhancer is added in an amount of 0.01 to 10% by weight based on the total mass of the anti-aging cream, for example: 0.5%, 1%, 2%, 3%, 5%, 6%, 7%, 8%, etc. When the addition amount of the permeation enhancer is less than 0.01%, the permeation enhancer effect is not obvious. When the addition amount of the permeation enhancer is more than 10%, the permeation enhancer cannot further act.

Preferably, in the present application, the anti-aging cream further comprises one or a combination of two or more of oils, emulsifiers, moisturizers, rheology modifiers, antioxidants, skin conditioners, chelators, preservatives, fragrances, PH modifiers. The adding amount of the grease is 4-15% based on the total mass of the anti-aging face cream; the addition amount of the emulsifying agent is 2-4%; the addition amount of the humectant is 0.01-20%; the addition amount of the rheological modifier is 0.2-1.8%; the addition amount of the antioxidant is 0.01-1%; the addition amount of the skin conditioning agent is 0.01-10%; the addition amount of the chelating agent is 0.01-1%; the addition amount of the preservative is 0.01-1.5%; the addition amount of the aromatic is 0.005-0.5%.

The formula of the anti-aging face cream is mild, so that the efficacy of the collagenase inhibitor composition prepared by the licorice root extract and the chamomile extract can be fully exerted.

Specifically:

the grease generally forms an oil film in the cleaning product to regulate skin feel and play a role in smoothing hair, and supplements grease for skin and hair in the nursing product to play a role in nursing. The oils and fats are generally classified into three types, mineral oils and fats, synthetic oils and fats, and natural oils and fats.

Preferably, the anti-aging cream according to the present application, the oil comprises one or a combination of two or more of white oil, vaseline, lanolin derivative, silicone oil, higher fatty acid ester, natural vegetable oil, natural animal oil, and higher fatty alcohol.

The amount of the fat added is 4 to 15% by weight of the total mass of the anti-aging cream, and for example, the amount of the fat added may be 4%, 6%, 8%, 10%, 12%, 15%. The grease can bring different greasing and moisturizing performances compared with grease types, and part of grease has certain fluidity and has great response to skin feel. The oil is the basic component of the anti-aging face cream. In addition, by adding the grease, a hydrophobic film can be formed on the skin surface, and invasion of external harmful substances can be prevented. The cream products need a large amount of grease to support, so the content of the grease is more than 4%; however, the content of grease is more than 15%, so that the anti-aging cream is too greasy, and the use feeling is reduced.

The emulsifier can be divided into three types according to the action mechanism, the first type is represented by surfactant, the surfactant is adsorbed on the oil-water interface, the interfacial tension of oil-water is reduced, and the increase of the total interfacial energy of the system caused by the reduction of dispersed phase particles is further reduced. The second type is represented by a high molecular polymer which has a steric stabilization effect, namely contains hydrophilic groups and lipophilic groups, is generally adsorbed at an acute-nonpolar interface, part of chain links of the polymer can go deep into two phases to form an effective liquid bead protection layer, and the third type is represented by solid particles, wherein highly dispersed solid particle powder has large surface energy, so that the polymer has certain surface activity and can be adsorbed on an oil-water interface.

Preferably, the anti-ageing face cream according to the application, the emulsifier comprises one or a combination of two or more of cetostearyl alcohol (and) cetostearyl alcohol polyether-20, behenyl alcohol polyether-25, cetostearyl alcohol, cetostearyl ether-30, stearyl alcohol polyether-21, stearyl alcohol polyether-2, cetostearyl alcohol and coco glucoside, sorbitan monolaurate, sorbitan monooleate, polyoxyethylene (20) monopalmitic acid.

In the present application, the amount of the emulsifier added is 2 to 4% based on the total mass of the anti-aging cream, due to the presence of a large amount of grease in the system, for example: may be 2.0 to 2.5%, 3 to 3.6%, etc. When the dosage of the emulsifier is less than 2%, insufficient emulsification is caused, so that the system is unstable; when the amount of the emulsifier is more than 4%, the stability of the product is also affected to some extent.

The moisturizing agent can impart moisturizing property to the cream, and is generally classified into polyalcohols, high molecular biological polysaccharides, natural moisturizing factors and others, including ceramide, urea and the like. The moisturizing agents are compounded generally, so that the moisturizing performance of the moisturizing agents is better exerted.

Preferably, the anti-aging facial cream is prepared by compounding a plurality of humectants, wherein the humectant comprises one or more than two of glycerin, sorbitol, bioglycol-1, dipropylene glycol, glycerol, 1, 3-butanediol, propylene glycol, polyethylene glycol, sodium hyaluronate and ceramide.

The moisturizing agent is added in an amount of 0.01-20% based on the total mass of the anti-aging face cream, and can play a role in moisturizing and supplementing water. In order to further exert the effect of the humectant, the humectant of the present application may be added in an amount of 1 to 18%, 2 to 16%, 3 to 14%, 4 to 15%, 5 to 14%, 6 to 13%, or 7 to 12%. When the content of the humectant is less than 0.01%, the moisturizing effect is not obvious; when the content of the humectant is higher than 20%, the anti-aging facial cream has sticky feel.

Rheology modifiers in cream-like systems generally rely on synthetic water-soluble polymers to increase viscosity by increasing viscosity of the external phase, the rheology modifiers of the present application acting to thicken. Different types of rheology modifiers have a large difference in the impact on the cream system, for example, natural water-soluble polymers (xanthan gum, guar gum, etc.), semisynthetic water-soluble polymers (polyethyl cellulose) mainly play a role in suspension, do not greatly increase the viscosity of the cream, while synthetic water-soluble polymers mostly have a significant impact on the viscosity of the system.

Preferably, the anti-aging cream according to the present application, wherein the rheology modifier comprises one or a combination of two or more of ammonium acryloyldimethyl taurate/VP copolymer, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, acrylic acid (esters) type/C10-30 alkanol acrylate cross-linked polymer, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer and a complex of isohexadecane and polysorbate-60.

The rheology modifier is added in an amount of 0.2 to 1.8% by total mass of the anti-aging cream, for example, the rheology modifier may be added in an amount of 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, etc. When the addition amount of the rheology modifier is between 0.2 and 1.8 percent, the adhesive has low sticky feel, excellent use feel, good dispersibility and quick absorption. When the addition amount of the rheology modifier is less than 0.02%, the anti-aging facial cream has thinner texture and no sticky feeling; when the addition amount of the rheology modifier is more than 1.8%, the anti-aging cream is too heavy, which increases the burden on the skin.

The anti-aging cream is added with antioxidant, can improve antioxidant capacity, and can prevent oily components such as oil, wax, hydrocarbon, etc. of cosmetic from contacting oxygen in air to generate oxidation, and generate peroxide, aldehyde, acid, etc., so as to make cosmetic color change, rancidity, quality degradation, etc.

Preferably, the antioxidant comprises one or more of vitamin E, VE acetate, 2, 6-di-tert-butyl-4-methylphenol, lycopene, ascorbyl ethyl ether and the like.

The addition amount of the antioxidant is 0.01-5% based on the total mass of the anti-aging face cream; when the amount of the additive is less than 0.01%, the antioxidation effect cannot be exerted, and when the amount of the antioxidant is more than 2%, the antioxidation effect cannot be increased any more, and the cost is increased.

Preferably, the anti-aging cream further comprises a skin conditioner, wherein the skin conditioner comprises one or more than two of allantoin, D-panthenol, hydrolyzed collagen, oat peptide, ceramide 3, fucus vesiculosus extract, chlorella fermentation product, chlorella extract, brown algae extract, hamamelis mollis water, bisabolol, allantoin, ginkgo mistletoe leaf extract, cogongrass rhizome extract, serine, kelp extract, beta-glucan and cactus extract.

The addition amount of the skin conditioning agent is 0.01-10% based on the total mass of the anti-aging face cream; the skin conditioner is added to calm the skin, so that the facial skin injury red swelling can be relieved to a certain extent, and the generation of wrinkles can be reduced.

The anti-aging face cream of the present application may further be appropriately added with a chelating agent. The chelating agent is added in an amount of 0.01-1% based on the total mass of the anti-aging face cream. The chelating agent may be disodium EDTA and/or tetrasodium EDTA, and the like.

Preferably, the anti-aging cream of the present application further comprises a preservative and a fragrance. The preservative in the anti-aging face cream is added in an amount of 0.01-1.5% by weight of the total mass of the anti-aging face cream, and the aromatic is added in an amount of 0.005-0.5%.

Preferably, the preservative may include one or a combination of two or more of phenoxyethanol, methylparaben, propylparaben, benzoic acid and salts thereof. The aromatic may be essence, etc.

Preferably, the anti-aging mask further comprises a PH regulator, wherein the PH regulator is used for regulating the PH value to be between 5.5 and 6.5. The pH value of the anti-aging face cream is more suitable for human skin.

Preferably, the pH regulator comprises one or more than two of citric acid, tartaric acid, phosphoric acid or sodium hydroxide, potassium hydroxide and triethanolamine.

As a third aspect of the present application, there is also provided a method of preparing an anti-aging cream comprising the step of mixing the components of the anti-aging cream.

Specifically, the anti-aging facial cream is determined as the following process according to compatibility, freezing point and high-low temperature stability, and comprises the following steps:

1. respectively adding the emulsifier, the grease, the humectant, the penetration enhancer and part of water into an oil phase beaker and a water phase beaker, stirring and heating to 80-85 ℃, and respectively preserving heat at 70-75 ℃ after the emulsifier, the grease, the humectant, the penetration enhancer and part of water are uniformly dissolved;

2. the oil phase beaker is kept at 70-75 ℃, the oil phase is homogenized, the water phase which is uniformly dispersed in advance is added, the homogenization is carried out for 5min, and the heat preservation and stirring are carried out for 30min;

3. the obtained material is kept at 70-75 ℃ and is slowly added with skin conditioning agent and rheology modifier which are evenly dissolved under the low-speed homogenization condition, and then is homogenized for 10min at high speed, and the emulsification temperature is controlled at 70-75 ℃;

4. after emulsification, placing the beaker into cold water, slowly stirring and cooling to 40-45 ℃, adding preservative and aromatic, homogenizing at high speed for 1min, finally adding licorice root extract and chamomile extract, adjusting the PH to a proper range by a proper amount of PH regulator, and supplementing the rest with water;

5. continuously stirring and cooling to room temperature;

6. discharging after inspection, and standing for 12-24 hours to obtain the anti-aging face cream.

The application has the beneficial effects that:

the collagenase inhibitor composition of the application has a synergistic effect by matching the licorice root extract and the chamomile extract, and has excellent effect of inhibiting collagenase activity.

The anti-aging face cream comprises the collagenase inhibitor composition and the penetration enhancer, can reduce collagenase activity, can reduce collagen degradation, plays an anti-aging role, has an excellent moisturizing effect, and has no side effect on human bodies.

The preparation method of the anti-aging face cream provided by the application is simple to operate, raw materials are easy to obtain, and the face cream can be used for mass production.

Drawings

FIG. 1 is a graph of sample concentration of licorice root extract versus collagenase inhibition rate, wherein the sample concentration is the mass concentration of licorice root extract;

FIG. 2 is a schematic diagram showing the results of measurement of the inhibition mechanism of the collagenase activity by the Glycyrrhiza glabra root extract;

FIG. 3 is a graph showing the results of a type of inhibition assay of collagenase activity by Glycyrrhiza glabra root extract;

FIG. 4 is a schematic representation of the results of collagenase activity inhibition by a collagenase inhibitor composition;

FIG. 5 is a graph showing the comparison of the skin elasticity change rates of application examples 1 to 5 of the present application and application comparative examples 1 to 5.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In the examples/comparative examples of the present application, the preparation method of the licorice root extract used was:

cleaning Glycyrrhrizae radix, oven drying, pulverizing, adding into extraction tank, adding water and ethanol at 45deg.C, wherein the mass ratio of Glycyrrhrizae radix raw material, water and ethanol is 1:1:2, and decocting for 3 times each for 2.5 hr to obtain Glycyrrhrizae radix extractive solution. Filtering the obtained licorice root extract, storing in a storage tank, allowing filtrate in the storage tank to slowly flow into an external circulation tank and a sedimentation tank for concentration to obtain a liquid component and a precipitate, spray-drying the liquid component, drying the precipitate, sieving with a 80-100-mesh sieve to obtain a licorice root extract, detecting the licorice root extract, wherein the ash content is 6%, and the water content is 4%.

In the inventive examples/comparative examples, chamomile extracts were purchased from: and is available from the state market exhibition macro biotechnology limited company.

Examples 1 to 5

The licorice root extract was diluted with deionized water to obtain 5 sets of test solutions of different concentrations (final concentrations). The mass concentration of the licorice root extract when tested for collagenase inhibition activity is shown in table 1 below.

Assay for inhibition of collagenase Activity

The Fu Lin Fen reagent can be reduced to blue by phenols (molybdenum blue and tungsten blue mixture) under alkaline condition, and can be used for determining protein and its hydrolysate due to amino acids containing phenol group (such as tyrosine, tryptophan, etc.) in protein moleculeProtease activity. Usually, casein is used as substrate, reacted with enzyme solution under certain pH value and temperature, after a period of time, the enzymatic reaction is stopped by trichloroacetic acid, after casein precipitate is removed by centrifugation or filtration, the supernatant is obtained, and NaOH and Na are used ₂ CO ₃ Alkalizing, adding Fu Lin Fen reagent to develop color, and measuring the blue color in proportion to the amount of polypeptide and tyrosine produced in the filtrate at 650nm wavelength with spectrophotometer to calculate the activity of proteinase.

Collagenase activity is measured as collagenase activity that catalyzes the production of amino acids from casein. The method comprises the following steps:

taking 1 test tube, adding deionized water 0.25mL and collagenase 0.25mL (32U/mL) respectively, then adding substrate casein solution 0.5mL (1.0%) and immediately mixing, carrying out water bath heat preservation at 37 ℃ for 10min, then adding trichloroacetic acid solution 1mL (6.5%) and mixing uniformly, standing for 10min, centrifuging at 10000rpm for 5min, and obtaining experimental supernatant 1.

Taking 1 test tube, adding 0.25mL of deionized water and 0.25mL of collagenase (32U/mL) respectively, then adding 1mL (6.5%) of trichloroacetic acid solution, immediately mixing, carrying out water bath heat preservation at 37 ℃ for 10min, then adding 0.5mL (1.0%) of substrate casein solution, mixing, standing for 10min, centrifuging at 10000rpm for 5min, and obtaining supernatant 2 of a control group.

Collagenase 0.25mL (32U/mL) was added to each of the 5 test tubes, then 0.25mL of the test solutions of examples 1 to 5 were added and mixed uniformly, then 0.5mL (1.0%) of the substrate casein solution was added and mixed uniformly immediately, after incubation in a water bath at 37℃for 10 minutes, then 1mL (6.5%) of trichloroacetic acid solution was added and mixed uniformly, and after 10 minutes, centrifugation was performed at 10000rpm for 5 minutes to obtain experimental group supernatant 3.

Collagenase 0.25mL (32U/mL) was added to each of the 5 test tubes, then 0.25mL of the test solutions of examples 1 to 5 were added and mixed uniformly, then 1mL (6.5%) of trichloroacetic acid solution was added and mixed uniformly immediately, after incubation in a water bath at 37℃for 10min, then 0.5mL (1.0%) of substrate casein solution was added and mixed uniformly, and after 10min, centrifugation at 10000rpm was performed for 5min to obtain control supernatant 4.

Taking 0.5mL of supernatant fluid sample, respectively adding 0.5mL of alkaline copper test solution (solution A, 10g of sodium hydroxide, 50g of sodium carbonate and 400mL of water to fully dissolve the sample, solution B, taking 0.5g of potassium tartrate, 50mL of water to dissolve the sample, taking 0.25g of copper sulfate, 30mL of water to dissolve the sample, and mixing the two solutions to obtain a solution B, namely, an alkaline copper reagent, namely, mixing the solution A, the solution B and the water according to the proportion of 20:4:1), and shaking the solution B uniformly. 2.0ml of Fu Lin Fen test solution (diluted 8 times of Fu Lin Fen test solution with 1mol/L acid concentration) is added after 10 minutes, and the mixture is immediately mixed; the sample was left at room temperature for 30 minutes, and absorbance was measured at a wavelength of 650 nm.

Inhibition ratio = [1- (A) ₃ -A ₄ )/(A _1- A ₂ )]×100％

Wherein: a is that ₁ Absorbance of the experimental group without adding licorice root extract;

A ₂ absorbance of control group without licorice root extract;

A ₃ absorbance of the experimental group for the extract containing licorice root;

A ₄ the absorbance of the control group contains licorice root extract.

TABLE 1

The results of the inhibition ratio on the abscissa and the ordinate of the mass concentration of the licorice root extract show that the licorice root extract has an excellent inhibition effect on collagenase activity based on table 1 and fig. 1, and the inhibition ratio on collagenase gradually increases as the mass concentration of the licorice root extract increases.

Comparative example 1

Deionized water was taken as the test solution for comparative example 1. Wherein, when the inhibition mechanism of collagenase activity was measured, the mass concentration of the licorice root extract was 0mg/L (shown in Table 2).

Examples 6 to 10

The licorice root extract was dissolved in 5 volumes of deionized water corresponding to comparative example 1 to obtain 5 sets of test solutions of different concentrations. The mass concentration of the licorice root extract in the measurement of the inhibition mechanism for inhibiting collagenase activity is shown in Table 2 below.

Inhibition mechanism determination of inhibition of collagenase activity by licorice root extract

To 30 test tubes, 0.25mL of the test solutions of comparative example 1 and examples 6 to 10 were added, and 0.05mL, 0.1mL, 0.15mL, 0.2mL, 0.25mL of collagenase (32U/mL) were added, and all of the D-PBS buffer having a pH of 7.4 was added to a final volume of 0.5mL, followed by 0.5mL (1.0%) of the substrate casein solution and immediately mixing, after 10 minutes of incubation in a water bath at 37℃1mL (6.5%) of trichloroacetic acid solution was added, mixing, and after 10 minutes of standing, centrifugation was performed at 10000rpm for 5 minutes to obtain a supernatant M of the test group.

To 30 test tubes, 0.25mL of the test solutions of comparative example 1 and examples 6 to 10 were added, and 0.05mL, 0.1mL, 0.15mL, 0.2mL, 0.25mL of collagenase (32U/mL) were added, and all of the D-PBS buffer having a pH of 7.4 was added to a final volume of 0.5mL, followed by 1mL (6.5%) of trichloroacetic acid solution and immediately mixing, and after incubation in a water bath at 37℃for 10 minutes, 0.5mL (1.0%) of the substrate casein solution was added, and mixing was performed, and after 10 minutes of standing, centrifugation was performed at 10000rpm for 5 minutes to obtain a supernatant N of the control group.

The absorbance A of the reaction solution M and the reaction solution N at 650nm was measured by the Fu Lin Fen method, and delta was calculated according to the following formula _1A650 The specific results are shown in table 2 below:

Δ _1A650 ＝A _M -A _N

wherein A is _M Absorbance for the experimental group;

A _N absorbance was used as control.

Then, the initial reaction rate (absorbance change value. DELTA.) of the enzyme was measured on the abscissa of the collagenase concentration (U/mL) _1A650 ) The change curves of the collagenase concentration-absorbance of comparative example 1 and example 9 were made on the ordinate, and the results are shown in fig. 2.

Specifically, if the change curve of the concentration-absorbance of collagenase of examples 6-10 intersects the change curve of the concentration-absorbance of collagenase of comparative example 1 at the origin, it is determined that the reversibility is suppressed; an irreversible inhibition was determined if the concentration-absorbance curve of collagenase of examples 6-10 was parallel to the concentration-absorbance curve of collagenase of comparative example 1 and did not pass through the origin.

TABLE 2

As can be seen from table 2 and fig. 2, the licorice root extract has reversibility in inhibiting collagenase activity.

Comparative example 2

Deionized water was taken as the test solution for comparative example 2. Wherein, when the inhibition mechanism of collagenase activity was measured, the mass concentration of the licorice root extract was 0mg/L (shown in Table 3).

Examples 11 to 15

The licorice root extract was dissolved in 5 volumes of deionized water corresponding to comparative example 1 to obtain 5 sets of test solutions of different concentrations. The mass concentration of the licorice root extract in the measurement of the inhibition mechanism for inhibiting collagenase activity is shown in Table 3 below.

Inhibition type assay of Glycyrrhiza uralensis root extract on collagenase

0.5mL, 0.4mL, 0.3mL, 0.2mL, 0.1mL of substrate casein solution (1.0%) was added to 30 test tubes, tris HCl buffer with pH of 7.4 was added to a final volume of 0.5mL, then 0.25mL of the test solutions of comparative example 2 and examples 11 to 15 were added, followed by collagenase (32U/mL) and immediately mixing, after incubation in a 37℃water bath for 10min, 1mL (6.5%) of trichloroacetic acid solution was then added, mixing was performed, and after 10min standing, centrifugation was performed at 10000rpm for 5min to obtain control supernatant m.

0.5mL, 0.4mL, 0.3mL, 0.2mL, 0.1mL of substrate casein solution (1.0%) was added to 30 test tubes, tris-HCl buffer solution with pH of 7.4 was added to a final volume of 0.5mL, then 0.25mL of the test solutions of comparative example 2 and examples 11-15 were added, 1mL (6.5%) of trichloroacetic acid solution was added and immediately mixed, after incubation in a 37℃water bath for 10min, collagenase (32U/mL) was added and mixed, and after 10min standing, centrifugation was performed at 10000rpm for 5min to obtain supernatant n of the control group.

Determination of absorbance A at 650nm of the above-mentioned reaction solution m and reaction solution n by Fu Lin Fen method _m And A _n Delta is calculated according to the following formula _2A650 The specific results are shown in table 3 below:

Δ _2A650 ＝A _m -A _n

wherein A is _m Absorbance for the experimental group;

A _n absorbance was used as control.

The results are shown in Table 3. The inverse of the substrate concentration is taken as the abscissa, and the initial reaction rate of the enzyme (change in absorbance delta _2A650 ) The reciprocal of (2) is a reciprocal double reciprocal curve of the reciprocal of the substrate concentration as the reciprocal of the change in absorbance, see FIG. 3.

TABLE 3 Table 3

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As can be seen from table 3 and fig. 3, the double reciprocal curves of examples 11 to 15 intersect the double reciprocal curve of comparative example 1 at the abscissa, and the intercept of the ordinate becomes larger as the collagenase inhibitor concentration increases, it can be judged that the inhibition of collagenase activity by the licorice root extract is a non-competitive inhibition.

Comparative examples 3 to 4

The licorice root extract was taken as the test solution of comparative example 3 with a mass fraction of 4%. The chamomile extract was taken as the test solution of comparative example 4 with a mass fraction of 4%.

Examples 16 to 20

The extract of Glycyrrhrizae radix and flos Matricariae Chamomillae is used as collagenase inhibitor. The licorice root extract and the chamomile extract were mixed in mass ratios of 1:1 (example 16), 3:2 (example 17), 2:3 (example 18), 9:1 (example 19), 1:9 (example 20), to obtain a collagenase inhibitor composition having a total concentration of 4%. Determination of collagenase Activity inhibition by combination of Glycyrrhiza root extract and Chamomile extract

Taking 1 test tube, adding deionized water 0.25mL and collagenase 0.25mL (32U/mL) respectively, then adding substrate casein solution 0.5mL (1.0%) and immediately mixing, carrying out water bath heat preservation at 37 ℃ for 10min, then adding trichloroacetic acid solution 1mL (6.5%) and mixing uniformly, standing for 10min, centrifuging at 10000rpm for 5min, and obtaining experimental supernatant 5.

Taking 1 test tube, adding 0.25mL of deionized water and 0.25mL of collagenase (32U/mL) respectively, then adding 1mL (6.5%) of trichloroacetic acid solution, immediately mixing, carrying out water bath heat preservation at 37 ℃ for 10min, then adding 0.5mL (1.0%) of substrate casein solution, mixing, standing for 10min, centrifuging at 10000rpm for 5min, and obtaining supernatant 6 of a control group.

0.25mL of the test solutions of comparative examples 3-4 and examples 15-19 were added to 7 test tubes, 0.25mL of collagenase (32U/mL) was added, and D-PBS buffer having pH of 7.4 was added to the whole to a final volume of 0.5mL, followed by addition of 0.5mL (1.0%) of substrate casein solution and immediate mixing, incubation in a water bath at 37℃for 10min, 1mL (6.5%) of trichloroacetic acid solution was added and mixed, and after 10min standing, centrifugation at 10000rpm for 5min, to give supernatant 7 of the test group.

To 7 test tubes, 0.25mL of the test solutions of comparative examples 3 to 4 and examples 15 to 19 were added, and 0.25mL of collagenase (32U/mL) was added, and D-PBS buffer having pH of 7.4 was added to a final volume of 0.5mL, followed by 1mL (6.5%) of trichloroacetic acid solution and immediately mixing, and after 10 minutes of incubation in a water bath at 37℃0.5mL (1.0%) of substrate casein solution was added and mixed, and after 10 minutes of standing, centrifugation was performed at 10000rpm for 5 minutes to obtain supernatant 8 of the control group.

The absorbance A of the reaction solutions 5, 6, 7 and 8 at 650nm was measured by the Fu Lin Fen method.

Inhibition ratio = [1- (A7-A8)/(A5-A6) ] ×100%

Wherein: a5 is absorbance of the experimental group without adding licorice root extract and chamomile extract;

a6 is absorbance of the control group without adding licorice root extract and chamomile extract;

a7 is absorbance of an experimental group containing licorice root extract and/or chamomile extract;

a8 is absorbance of control group containing Glycyrrhrizae radix extract and/or flos Matricariae Chamomillae extract.

TABLE 4 Table 4

As can be seen from table 4 above, under the condition that the total concentration of collagenase inhibitor is 4mg/mL, compared with comparative example 3 and comparative example 4 which use licorice root extract or chamomile extract alone, the synergistic effect is achieved by adopting the combination of the two extracts, the inhibition rate can be improved, and when the mass ratio of chamomile extract to licorice root extract is 1:1-9, the inhibition effect is better, and when the mass ratio of chamomile extract to licorice root extract is 1:1 or 1:9, wherein the mass ratio of chamomile extract to licorice root extract is 1: the best value is 1.

Application examples 1 to 5

According to the contents (mass percent) of each component in the formulation of the anti-aging creams of application examples 1 to 5 in tables 4 to 5 below, an anti-aging cream was prepared according to the following production process steps. The production process comprises the following steps:

1. adding the phase A and the phase B into an oil phase beaker and a water phase beaker respectively, stirring and heating to 80-85 ℃, and respectively preserving heat at 70-75 ℃ after the phases are uniformly dissolved;

2. keeping the temperature of the phase A at 70-75 ℃, homogenizing the phase A, adding the phase B which is uniformly dispersed in advance, and homogenizing for 5min;

3. the obtained material body is kept at 70-75 ℃ and is slowly added with the evenly dissolved C phase under the low-speed homogenization condition;

4. after emulsification is finished, placing the beaker into cold water, slowly stirring and cooling to 40-45 ℃, adding the D phase, homogenizing at a high speed for 1min, and finally adding the E phase;

5. continuously stirring and cooling to room temperature;

6. discharging after the inspection is qualified, and standing for 12-24 hours;

7. and after the inspection is qualified, sub-packaging and packaging, inspecting again, and warehousing the finished product.

Note that: a, B, C, D, E phases in the process are respectively

Phase A: cetyl stearyl alcohol and coco glucoside, stearyl polyether-21, stearyl polyether-2, cetostearyl alcohol, synthetic squalane, caprylic/capric triglyceride, dioctyl carbonate, butter fruit, and VE acetate;

and B phase: water, allantoin, butanediol, glycerol, sodium chloride, EDTA-Na2, xylitol, betaine, sodium hyaluronate, D-panthenol, bis-diethoxydiglycol cyclohexane 1, 4-dicarboxylate;

and C phase: sodium hyaluronate, hydroxyethyl acrylate, sodium acryloyldimethyl taurate copolymer, sorbitan isostearate, polysorbate-60, water, glycerol, acrylic acid (esters) and/or C10-30 alkanol acrylate cross-linked polymer;

and D phase: phenoxyethanol, methylparaben, essence and PEG-40;

e phase: water, sodium citrate, sodium pyrrolidone carboxylate, licorice root extract, chamomile extract;

in the formula, cetostearyl alcohol, synthetic squalane, caprylic acid triglyceride, dioctyl carbonate and butter fruit tree fruit fat are grease;

cetyl stearyl alcohol and coco glucoside, stearyl polyether-21, stearyl polyether-2 are emulsifiers;

butanediol, glycerol, xylitol, betaine, sodium hyaluronate, sodium pyrrolidone carboxylate are humectants;

bis-diethoxydiglycol cyclohexane 1, 4-dicarboxylate is a permeation enhancer;

VE acetate is an antioxidant; EDTA-Na2 is a chelator; phenoxyethanol and methylparaben are used as preservatives; the essence is a fragrance.

Hydroxyethyl acrylate, sodium acryloyldimethyl taurate copolymer, sorbitan isostearate, polysorbate-60, acrylic acid (esters) and C10-30 alkanol acrylate cross-linked polymer are rheology modifiers;

allantoin and D-panthenol are skin conditioners, and have the effects of nourishing and promoting wound healing; sodium chloride can improve the emulsifying property in the system.

TABLE 5 application examples 1-5

Comparative examples 1 to 5 were used

According to the contents (mass percentages) of the respective components in the cream formulations of application comparative examples 1 to 5 in the following table 6, creams were prepared in the same manner as in application examples 1 to 5.

Table 6 comparative examples 1-5 were used

Anti-aging efficacy test

Skin elasticity test method: the test principle is based on suction and stretching, and the suction pressure generated on the surface of the tested skin sucks the skin into a specific test probe, and the depth of the skin sucked into the test probe is measured by a non-contact optical test system. The test probe includes a light emitter and a light receiver, and the ratio of light (the ratio of emitted light to received light) is proportional to the depth of skin drawn into the probe, thus obtaining a plot of the length of skin stretched versus time. Measuring skin elasticity of a subject by using a probe PVM600 of German CK company and a skin elasticity measuring instrument MPA580, selecting a parameter R2 as a comparison index (R2: the ratio of the skin rebound amount without negative pressure to the maximum stretching amount with negative pressure is closer to 1, the better the skin elasticity is), measuring 3 times in total, and taking an average value; the improvement of skin elasticity in the test area by the product was evaluated by measuring the change in skin elasticity value R2 before and after use of the product.

The number of subjects was 33, the test period was 8 weeks, the anti-aging creams of application examples 1 to 5 and the creams of comparative examples 1 to 5 were selected for test, and applied to different areas on the inner side of the forearm in the morning and evening each day, and skin elasticity of the tested areas before and at 8 weeks of use was measured, respectively, to thereby characterize the rate of change of skin elasticity, and the results of the rate of change of specific elasticity are shown in fig. 4.

As can be seen from fig. 5, the application examples 1 to 5 of the present application have a large rate of change in elasticity, that is, skin elasticity is enhanced, and thus, the use of collagenase inhibitors in combination of licorice root extract and chamomile extract can effectively improve skin aging.

In the application of comparative examples 1 to 5 of the present application, when licorice root extract or chamomile extract was not used, the rate of change of skin elasticity was small and even the elasticity was weakened, and thus, the anti-aging effect was poor in the application of comparative examples 1 to 5. The combination of the licorice root extract and the chamomile extract has strong synergistic effect, can effectively inhibit collagenase activity, and has excellent anti-aging effect.

The above examples of the present application are merely illustrative of the present application and are not intended to limit the embodiments of the present application. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the application are desired to be protected by the following claims.

Claims

1. The collagenase inhibitor composition is characterized by comprising 50-99% of licorice root extract and 1-50% of chamomile extract by mass fraction, wherein the preparation method of the licorice root extract comprises the following steps:

step one: cleaning Glycyrrhrizae radix, oven drying, and pulverizing;

step two: adding the crushed licorice root raw material into an extraction tank, and extracting by using water and alcohol, wherein the mass ratio of the licorice root raw material to the water to the alcohol is 1:0.1 to 1: 0.5-2.5, the extraction temperature is 30-60 ℃,

extracting for 2-4 times for 2-8 hours to obtain licorice root extract;

2. The collagenase inhibitor composition according to claim 1, wherein the mass ratio of said chamomile extract to said licorice root extract is 1:1 to 9.

3. The anti-aging face cream is characterized by comprising a collagenase inhibitor composition and a penetration enhancer, wherein the addition amount of the collagenase inhibitor composition is 0.01-10% based on the total mass of the anti-aging face cream, the addition amount of the penetration enhancer is 0.01-10%, and the collagenase inhibitor composition consists of 50-99% of licorice root extract and 1-50% of chamomile extract based on mass fraction; the penetration enhancer is bis-diethoxy diglycol cyclohexane 1, 4-dicarboxylic acid ester, wherein the preparation method of the licorice root extract comprises the following steps:

step one: cleaning Glycyrrhrizae radix, oven drying, and pulverizing;

extracting for 2-4 times for 2-8 hours to obtain licorice root extract;

4. The anti-aging cream of claim 3, further comprising one or a combination of two or more of a grease, an emulsifier, a humectant, a rheology modifier, an antioxidant, a skin conditioner, a chelating agent, a fragrance, a preservative; the adding amount of the grease is 4-15% based on the total mass of the anti-aging face cream; the addition amount of the emulsifying agent is 2-4%; the addition amount of the humectant is 0.01-20%; the addition amount of the rheological modifier is 0.2-1.8%; the addition amount of the antioxidant is 0.01-1%; the addition amount of the skin conditioning agent is 0.01-10%; the addition amount of the chelating agent is 0.01-1%; the addition amount of the preservative is 0.01-1.5%; the addition amount of the aromatic is 0.005-0.5%.

5. The anti-aging cream of claim 4, wherein the oil comprises one or a combination of two or more of white oil, vaseline, lanolin derivative, silicone oil, higher fatty acid ester, natural vegetable oil, natural animal oil, and higher fatty alcohol; and/or the emulsifier comprises (and) cetostearyl alcohol, and one or a combination of more than two of cetostearyl alcohol polyether-20, behenyl alcohol polyether-25, cetostearyl alcohol, cetostearyl ether-30, steareth-21, steareth-2, cetostearyl alcohol and coco glucoside, sorbitan monolaurate, sorbitan monooleate, polyoxyethylene (20) monopalmitic acid.

6. The anti-aging facial cream of claim 4, wherein the humectant comprises one or a combination of two or more of glycerin, sorbitol, biogel-1, dipropylene glycol, glycerol, 1, 3-butanediol, propylene glycol, polyethylene glycol, sodium hyaluronate, and ceramide; and/or the rheology modifier comprises one or more than two of an ammonium acryloyldimethyl taurate/VP copolymer, a hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, a cross-linked polymer of acrylic acid (esters) and C10-30 alkanol acrylate, a hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer and a compound of isohexadecane and polysorbate-60.

7. The anti-aging cream of claim 4, wherein the antioxidant comprises one or a combination of more than two of vitamin E, VE acetate, 2, 6-di-tert-butyl-4-methylphenol, lycopene, ascorbyl ethyl ether, and the like; and/or

The skin conditioner comprises one or more than two of allantoin, D-panthenol, hydrolyzed collagen, oat peptide, ceramide 3, fucus vesiculosus extract, chlorella fermentation product, chlorella vulgaris extract, brown algae extract, hamamelis virginiana water, bisabolol, allantoin, ginkgo mistletoe leaf extract, cogongrass rhizome extract, serine, kelp extract, beta-glucan and cactus extract.

8. The anti-aging cream of claim 4, further comprising a PH adjuster comprising one or a combination of two or more of citric acid, tartaric acid, phosphoric acid, sodium hydroxide, potassium hydroxide, triethanolamine, wherein the PH adjuster is used to adjust the PH to between 5.5 and 6.5.

9. A method of preparing an anti-aging cream according to any one of claims 3 to 8, comprising the step of mixing the components of the anti-aging cream.