CN117137837A - Eye cream containing pomegranate flower extract and preparation method thereof - Google Patents

Eye cream containing pomegranate flower extract and preparation method thereof Download PDF

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Publication number
CN117137837A
CN117137837A CN202311106610.5A CN202311106610A CN117137837A CN 117137837 A CN117137837 A CN 117137837A CN 202311106610 A CN202311106610 A CN 202311106610A CN 117137837 A CN117137837 A CN 117137837A
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eye cream
stirring
flower extract
extract
pomegranate flower
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裴永艳
吴依凯
黄樱淇
雷星雨
周凤玲
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Guangdong Pharmaceutical University
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Guangdong Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/005—Preparations for sensitive skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

The application provides an eye cream containing pomegranate flower extract and a preparation method thereof, wherein the eye cream consists of a composite plant extract, an antibacterial agent, an antioxidant, a functional auxiliary agent, deionized water and the like, wherein the composite plant extract consists of a pomegranate flower extract, a jojoba seed extract, a European Li Zi extract, a avocado fruit extract and a lavender flower extract, and the traditional anti-aging component is replaced by adding the natural plant extract such as the pomegranate flower extract into the eye cream.

Description

Eye cream containing pomegranate flower extract and preparation method thereof
Technical Field
The application belongs to the technical field of skin care products, and particularly relates to eye cream containing pomegranate flower extract and a preparation method thereof.
Background
The eye cream is a skin care cosmetic product for periocular skin, has the effects of improving skin moisturizing and barrier functions, can tighten the eye skin according to the functional components and active substances of different products, can relieve dark circles, improve wrinkles and fine lines, gradually attach importance to the anti-aging function of the eye cream in the current young people, has single anti-aging component, is mostly chemical synthetic substances, has small applicable crowd range and high allergy probability, has the anti-aging effect by using natural extracts, is not easy to cause anaphylactic reaction, and has strong eye irritation and needs to be improved by adding preservatives after using the natural extracts.
Disclosure of Invention
The application aims to solve the technical problems that the prior eye cream has single anti-aging component, small applicable crowd range, strong eye irritation, no preservative and short shelf life, and needs to be added with a preservative;
the second object of the application is to provide a preparation method of eye cream containing the pomegranate flower extract.
In order to achieve the first object, the present application adopts the following scheme:
an eye cream containing pomegranate flower extract comprises the following components in percentage by mass:
the composite plant extract comprises the following components in percentage by mass:
preferably, the pomegranate flower extract is PLUMPOleoactif.
In the practical implementation process, the synergistic effect of the avocado fruit and the European Li Zi extract is not suitable for people extremely sensitive to skin, but the synergistic effect of various plant extracts can reduce the skin irritation of the people extremely sensitive to skin, and the European Li Zi extract and the avocado fruit extract have the effect of relieving, so that the eye irritation of eye cream is reduced, and the compound plant extract has an excellent anti-aging effect.
The pomegranate flower extract has the following effects as eye cream:
firstly, the pomegranate flower extract is rich in various natural antioxidants such as vitamin C, vitamin E, polyphenol compounds and the like, the antioxidants can neutralize free radicals, reduce the damage of oxidative stress to the skin of eyes, the free radicals are one of the main factors causing skin aging, the use of the antioxidants can slow down the skin aging process, and the antioxidants are derived from nature, are purely natural extracts, have small irritation to eyes and are suitable for eye creams.
Secondly, the guava flower extract can promote the synthesis of collagen, a protein mainly constituting the skin structure, which helps to maintain the firmness and elasticity of the skin. With age, collagen synthesis gradually decreases, and skin becomes flaccid and wrinkled. By promoting collagen synthesis, the pomegranate flower extract can improve the firmness of the skin of eyes, reduce the generation of fine lines and wrinkles, and play a remarkable role in resisting aging.
The jojoba seed extract as eye cream has the following advantages:
first, jojoba seed extract is rich in various antioxidants such as vitamin E and flavonoids, and can effectively neutralize free radicals and reduce damage of oxidative stress to the skin of eyes. The antioxidant effect helps to retard the aging process of the ocular skin, reducing the appearance of fine lines, wrinkles and other signs of aging.
Secondly, the main component of the jojoba seed extract is liquid wax, which is very similar to natural grease of skin, so that the jojoba seed extract can be easily melted and absorbed when contacting with the skin, the eye cream can quickly permeate into the surface layer and the deep layer of the skin when in use, moisture and moisture are provided for the skin of the eyes, and meanwhile, thick oil films are prevented from being formed on the skin, so that the eye cream can not increase burden and uncomfortable feeling on the eye area when in use, and can not block pores, thus being suitable for people with various skin types.
Preferably, the antibacterial agent comprises the following components in percentage by mass:
0.05-0.6% of 1, 2-hexanediol;
0.15-0.9% of p-hydroxyacetophenone;
0.21-2% of lavender flower extract.
In practical implementation, 1, 2-hexanediol has the following advantages as an antibacterial agent,
firstly, the hexanediol is a water-soluble humectant, and has good moisturizing performance. The skin care agent can absorb moisture in air and lock the moisture in skin cells, so that the moisture balance of the eye skin is maintained, and the moisture evaporation is reduced. Meanwhile, the skin care cream has mild property, does not cause irritation or allergy, and is suitable for tender and weak skin of eyes.
Secondly, the hexanediol has lower molecular weight and higher solubility, has good permeability, and can permeate bacterial cell walls and membranes to enter the inside of bacteria. Once inside the bacterial cells, hexanediol interferes with the metabolic processes and intracellular balance of the bacteria, thereby producing bactericidal effects on the bacteria;
the hexanediol can absorb water and form hydrate, so that the water concentration in bacterial cells is reduced, and the hydration of the hexanediol can interfere with the normal metabolism and cell functions of the bacteria because the bacteria need water to maintain the normal physiological functions of the bacteria, thereby inhibiting the growth and reproduction of the bacteria;
the hexanediol and lavender flower extract can influence the enzyme activity in bacteria, so that the metabolic pathway and vital activity of the bacteria are interfered, and particularly, the hexanediol has strong inhibition capability on certain specific enzymes of the bacteria, so that the bacteria cannot normally perform necessary metabolic reactions, and the growth of the bacteria is inhibited.
Preferably, the antioxidant comprises the following components in percentage by mass:
0.5-1.5% of hydroxy pinacolin retinol;
0.5-1.5% of tocopherol;
0.5-3% of hydroxypropyl tetrahydropyran triol.
Preferably, the humectant comprises the following components in percentage by mass:
2-5% of squalane;
0.5-2% of dimethyl silicone oil;
avocado oil 0.5-2%.
Preferably, the softener is caprylic/capric triglyceride, the skin feel regulator is panthenol, and the pH regulator is triethanolamine.
Preferably, the emulsifier isMELLIFERA MB。
In the actual course of the implementation process, the process,MELLIFERA MB has the following advantages as one of the components of the eye cream:
in one of the two ways,MELLIFERA MB is polyglycerol-6 distearate (and) jojoba esters (and) polyglycerol-3 bee wax esters (and) cetyl alcohol, and the preparation process is formed by converting natural components through a green chemical process, so that the method accords with the green development concept of the application.
And two, a second step of, in the second step,MELLIFERA MB as a multifunctional emulsifier has immediate and long-acting relieving effect, can be better applied to the skin facing sensitive muscles, and improves the comfort level of the sensitive muscles.
Preferably, the preparation method of the eye cream containing the pomegranate flower extract comprises the following steps of:
step 101, sequentially adding cetostearyl alcohol and carbomer into a stirring kettle containing glycerol, and dispersing for 3-10min to completely dissolve under the condition that the stirring temperature is 60-80 ℃ and 800-1200rpm to obtain a pre-dispersion system;
step 102, sequentially adding 1, 2-propylene glycol, skin feel modifier, sodium hyaluronate and part of deionized water into the pre-dispersion system in step 101, and carrying out heat preservation and stirring for 10-20min under the condition that the stirring temperature is 80-90 ℃ and 1500-2200rpm to obtain a phase A;
step 103, sequentially adding the composite plant extract, the emulsifier, the 1, 3-propylene glycol, the rest deionized water, the antioxidant, the softener, the beeswax, the humectant and the antibacterial agent into a stirring kettle, and stirring for 5-10min at 60-80 ℃ and 1200-1800rpm to obtain a phase B;
and 104, sequentially adding the pH regulator, the phase A in the step 102, the phase B in the step 103 and the acetyl hexapeptide-8 into a stirring kettle, and stirring uniformly to obtain the eye cream.
Preferably, the stirring parameters in the step 104 are that stirring is carried out for 3-10min under the condition of 80-90 ℃ and 8000-8500rpm, after dispersing is finished, acetyl hexapeptide-8 is added after cooling to 35-40 ℃, and stirring is carried out for 8-15min under the condition of 1200-1500rpm, thus obtaining the eye cream.
More preferably, in step 102, the deionized water is partially 30-50% by volume, which is more beneficial to improving the overall dispersion efficiency.
Compared with the prior art, the application has the following advantages:
1. the application provides an eye cream containing pomegranate flower extract, which consists of a composite plant extract, an antibacterial agent, an antioxidant, a functional auxiliary agent and deionized water, wherein the composite plant extract consists of a pomegranate flower extract, a jojoba seed extract, a European Li Zi extract, a avocado fruit extract and a lavender flower extract, and the traditional anti-aging component is replaced by adding the natural plant extract such as the pomegranate flower extract into the eye cream.
2. The preparation method of the eye cream containing the pomegranate flower extract provided by the application is simple to operate, and the system is better dispersed by adding various materials after a pre-dispersion system is obtained, and the system contains a large amount of water, so that the eye cream is strong in adaptability to skin under eyes, applicable to production of skin care products without types, and has the advantage of being convenient to popularize and implement.
Drawings
FIG. 1 is a table of results of a Hacat cytotoxicity test on Hacat of an example of the Hacat cytotoxicity test of the present application;
FIG. 2 is a table showing the inhibition effect of the example on elastase in the anti-aging property test of the present application;
FIG. 3 is a table of toxicity test results of example HaCaT cells in the detection of antioxidant property according to the present application;
FIG. 4 is a table of results of Hacat cytotoxicity assays of examples and comparative examples in the detection of the protective effect of the present application on UVB-induced photoaged cells;
FIG. 5 is a comparison of chick embryo chorioallantoic membrane assay, positive and negative groups according to the present application.
Detailed Description
The application is further described in connection with Table 1, examples 1-3:
example 1.
The preparation of the eye cream containing the pomegranate flower extract is carried out according to the mass fraction table shown in table 1, and comprises the following steps:
step 101, sequentially adding glycerol, an emulsifying agent and carbomer into a stirring kettle, and dispersing for 3-10min under the condition that the stirring temperature is 60-80 ℃ and 800-1200rpm to obtain a pre-dispersion system;
step 102, sequentially adding propylene glycol, panthenol, sodium hyaluronate and part of deionized water into a stirring kettle, and stirring for 10-20min under the condition that the stirring temperature is 80-90 ℃ and 1500-2200rpm to obtain a phase A;
step 103, sequentially adding squalane, a composite plant extract, an emulsifying agent, propylene glycol, residual deionized water, tocopherol, hydroxypropyl tetrahydropyran triol, caprylic/capric triglyceride, beeswax, dimethyl silicone oil, p-hydroxyacetophenone and 1, 2-hexanediol into a stirring kettle, and stirring for 5-10min at 60-80 ℃ and 1200-1800rpm to obtain a phase B;
step 104, sequentially adding ethanolamine, A phase, B phase and acetyl hexapeptide-8 into a stirring kettle, homogenizing and stirring for 3-10min at 80-90 ℃ and 8000-8500rpm, cooling to 35-40 ℃ after dispersing, adding acetyl hexapeptide-8, and stirring for 8-15min at 1200-1500rpm to obtain the eye cream.
Example 2.
The preparation of the eye cream containing the pomegranate flower extract is carried out according to the mass fraction table shown in table 1, and comprises the following steps:
step 101, sequentially adding glycerol, an emulsifying agent and carbomer into a stirring kettle, and dispersing for 3-10min under the condition that the stirring temperature is 60-80 ℃ and 800-1200rpm to obtain a pre-dispersion system;
step 102, sequentially adding propylene glycol, panthenol, sodium hyaluronate and part of deionized water into a stirring kettle, and stirring for 10-20min under the condition that the stirring temperature is 80-90 ℃ and 1500-2200rpm to obtain a phase A;
step 103, sequentially adding squalane, a composite plant extract, an emulsifying agent, propylene glycol, residual deionized water, tocopherol, hydroxypropyl tetrahydropyran triol, caprylic/capric triglyceride, beeswax, dimethyl silicone oil, p-hydroxyacetophenone and 1, 2-hexanediol into a stirring kettle, and stirring for 5-10min at 60-80 ℃ and 1200-1800rpm to obtain a phase B;
step 104, sequentially adding ethanolamine, A phase, B phase and acetyl hexapeptide-8 into a stirring kettle, homogenizing and stirring for 3-10min at 80-90 ℃ and 8000-8500rpm, cooling to 35-40 ℃ after dispersing, adding acetyl hexapeptide-8, and stirring for 8-15min at 1200-1500rpm to obtain the eye cream.
Example 3.
The preparation of the eye cream containing the pomegranate flower extract is carried out according to the mass fraction table shown in table 1, and comprises the following steps:
step 101, sequentially adding glycerol, an emulsifying agent and carbomer into a stirring kettle, and dispersing for 3-10min under the condition that the stirring temperature is 60-80 ℃ and 800-1200rpm to obtain a pre-dispersion system;
step 102, sequentially adding propylene glycol, panthenol, sodium hyaluronate and part of deionized water into a stirring kettle, and stirring for 10-20min under the condition that the stirring temperature is 80-90 ℃ and 1500-2200rpm to obtain a phase A;
step 103, sequentially adding squalane, a composite plant extract, an emulsifying agent, propylene glycol, residual deionized water, tocopherol, hydroxypropyl tetrahydropyran triol, caprylic/capric triglyceride, beeswax, dimethyl silicone oil, p-hydroxyacetophenone and 1, 2-hexanediol into a stirring kettle, and stirring for 5-10min at 60-80 ℃ and 1200-1800rpm to obtain a phase B;
step 104, sequentially adding ethanolamine, A phase, B phase and acetyl hexapeptide-8 into a stirring kettle, homogenizing and stirring for 3-10min at 80-90 ℃ and 8000-8500rpm, cooling to 35-40 ℃ after dispersing, adding acetyl hexapeptide-8, and stirring for 8-15min at 1200-1500rpm to obtain the eye cream.
Table 1 examples 1-3 weight fraction tables of the respective components of eye creams containing Granati flower extract
The eye cream prepared in example 1 was tested as follows:
(1) And (3) detecting the corrosion resistance:
preparing test bacterial liquid:
A. standard strains are selected: selecting standard strains of staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, aspergillus niger and saccharomycetes;
B. standard bacteria activation: nutrient Agar (NA), sa agar culture medium (SDA) as culture medium, physiological saline as diluent, inoculating above standard strains into the culture medium, and culturing at 37deg.C for 4For 8 hours, the mold was incubated at 28℃for 72 hours (wherein the bacterial concentration was 5X 10 6 CFU/mL with mold concentration of 3X 10 5 CFU/mL)；
C. And (3) testing corrosion resistance: weighing 20g of eye cream samples prepared in example 1, respectively filling the eye cream samples into a sterilized container, respectively adding 0.2mL of staphylococcus aureus, escherichia coli and pseudomonas aeruginosa suspension, diluting to 0.2mL of aspergillus niger suspension and saccharomycete suspension, fully shaking and uniformly mixing, and using physiological saline as a neutralizing agent according to the ratio of sample liquid to neutralizing agent of 1: after 10 dilution, 1mL of sample dilution and 15mL of agar medium were plated, 10 in decibel at day O,7, 14 -2 Dilution, the bacteria content of the samples was counted using a dilution plate decantation method.
Detection result: as shown in table 2 below, the bacteria decreased by at least 99.9% within 7 days and did not increase within 28 days; the mould is reduced by at least 90% within 7 days and is not increased within 28 days, and the test method of preservative in composite ASTME640-06 (2019) aqueous cosmetics provides that the application has excellent antiseptic and antibacterial properties.
Table 2 antiseptic antibacterial test results table
(2) Hacat cytotoxicity assay:
the detection method comprises the following steps: determination of HacaT cell viability by MTT method, taking HacaT cells in logarithmic phase, digestion with pancreatin and use of 10% 5 Each ml was inoculated into a 96-well plate and incubated until the cells adhered to the wall.
Preparing a sample mother solution by using PBS, diluting the sample mother solution into concentration of 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.0078125% and 0.00390625% by weight by using DMEM culture medium, discarding old culture solutions in holes, respectively adding 100ul of sample solutions in each concentration in an experiment group, only replacing new DMEM culture medium in a control group, setting 6 compound holes in each concentration, placing the culture solution in an incubator for incubation for 24 hours, discarding old culture solution in the holes, carefully cleaning the culture solution by using PBS for one time, adding the prepared MTT solution in each hole in 100ul, placing the culture solution in the incubator for 4 hours, dissolving crystal formazan by using DMSO, measuring absorbance OD by using a 490nm wavelength of an enzyme-labeled instrument, and calculating the survival rate of cells according to the following formula: cell activity = [ (OD value of each well-blank)/(OD value of control-blank) ]x100%.
As shown in FIG. 1, it is clear from FIG. 1 that the HaCaT cell viability gradually decreases with increasing product concentration, and that generally, cell viability of more than 90% is considered to be non-toxic to cells.
(3) Eye irritation detection 1: (erythrocyte hemolysis experiment)
A. Measurement of the hemolysis Rate: taking 5 clean 10ml centrifuge tubes, wherein the tubes 1 to 5 are eye cream test groups, the tube 6 is a physiological saline solution as a negative control, the tube 7 is distilled water as a complete hemolysis control, and the tube 8 is a 100mg/L Sodium Dodecyl Sulfate (SDS) solution as a positive control.
Respectively sucking 2.5mL of erythrocyte suspension into a No. 1-7, no. 8 centrifuge tube, sequentially adding eye cream samples with different volumes into the No. 1-5 tube according to gradient concentration, fixing the volume to 5.0mL by using normal saline, and uniformly mixing to obtain eye cream tested samples with series concentration;
incubating the No. 1-8 tube in a constant temperature water bath at 37+ -0.5 ℃ for 0min, taking out to terminate the reaction, centrifuging at 3000rpm for 10min, absorbing supernatant, taking normal saline as blank control, measuring ultraviolet A values at wavelengths of 540, 560 and 575nm respectively, setting 3 parallel samples for each test object, and taking an average value.
When the wavelength is 560nm, the hemolysis rate [ HR, HR= (test sample group A560-negative control group A560)/(total hemolysis control group A560-negative control group A560) ] of the subject is calculated according to a formula, the mass concentration (C) of the eye cream test sample is taken as an abscissa, HR is taken as an ordinate, linear regression is performed, a regression equation HR=0.0262C-0.0115 is obtained, when HR=50%, the concentration of the sample solution which leads to 50% erythrocyte hemolysis is calculated according to the linear regression equation, namely the HC50 value is 1908mg/L (expressed in mg/L), and the result is reserved as an integer.
B. Measurement of hemoglobin denaturation index: and (3) taking the mass concentration (C) of the eye cream sample as an abscissa and the value A as an ordinate, and performing a linear regression equation. When the wavelength is 540nm, the linear equation A540=0.0157C-0.0051 is obtained; at a wavelength of 575nm, the linear equation a575=0.0146-0.013 is obtained.
HC50, namely concentration 1908mg/L, of the eye cream test sample is substituted into the linear regression equation, and A540 and A575 are calculated to be 29.95 and 27.84 respectively.
Calculating A575/A540 values of distilled water of the complete hemolysis control group, an eye cream tested sample and SDS solution supernatant of the positive control group respectively, and calculating hemoglobin denaturation indexes [ DI, DI= (R1-R2)/(R1-R3) of the eye cream tested sample according to a formula, wherein R1 is the A575/A540 value of the distilled water supernatant of the complete hemolysis control group, R2 is the A575/A540 value of the eye cream tested sample supernatant, and R3 is the A575/A540 value of the SDS solution supernatant of the positive control group ] is 13.44%.
Detection result:
based on HC50DI of the eye cream subject, HC50/DI (L/D) was calculated as 141.96, and the eye irritation classification criteria for the erythrocyte hemolysis test as shown in Table 3 below, since L/D > 100, showed that the eye cream prepared in example 1 was free from eye irritation.
TABLE 3 eye irritation fractionation criteria for erythrocyte hemolysis experiments
L/D Grading standard
L/D＞100 No irritation
10＜L/D≤100 Microstimulation
1＜L/D≤10 Mild irritation
0.1＜L/D≤1 Moderate irritation
L/D≤0.1 Severe irritation
(4) Eye irritation detection 2: (chick embryo chorioallantoic Membrane test)
A. Test article and preparation:
the eye cream prepared in example 1 is a cream-type test, and should be tested by using an endpoint evaluation method; the test is carried out as it is without dilution, the test is in the form of a paste, the test is coated on the surface of a plastic film (such as a sealing film) to form a thin layer, the thin layer is covered on a CAM film to enable the test to be in direct contact with the CAM film, the film is removed after the action time is over, and the test is carried out by adopting a reaction time method with a transparent solution with maximum solubility.
Negative control: sodium chloride solution with the mass concentration of 0.9% is selected for washing and negative control after the test object acts. A negative control of 0.9% sodium chloride is arranged for each test of the test object, so that the test condition can not cause the occurrence of a stimulating reaction;
positive control: 0.1mol/L sodium hydroxide solution, in order to evaluate the severity of ocular irritation of the test subjects.
B. Irritation detection:
for detecting solid test substances such as paste, 0.3ml of the eye cream paste prepared in the extruded example 1 is smeared on a sealing film to directly act on a CAM film, so that at least 50% of the surface of the CAM film is covered by the test substances, after 3min of the effect, the test substances on the CAM film are gently washed by normal saline, and the washing operation can mask slight bleeding change on the CAM film quickly, so that the result is observed after 30s of washing is completed, and the degree of each toxic effect change is observed. If the observation result shows that the score of at least 1 reaction of all 6 chick embryos is more than moderate (total score is not less than 12), the test should be repeated once.
To further understand the stimulation characteristics of solid and turbid stimulating substances in a dissolved state, the test is repeated once by using a transparent solution with the highest solubility of the test substance in water, the evaluation is performed by using a reaction time method, and the final result evaluation is based on the undiluted test substance source dosage form by using an end point method
C. Observation results: bleeding was graded and scored according to severity of bleeding, clotting, and vascular lysis.
The end point score (ES) should be calculated for the test performed using the end point evaluation method, with the result remaining two bits after the decimal point: score per chick embryo = sum of extent of bleeding, coagulation and vascular thawing observed per chick embryo; es=the average of the mathematical sums of 6 chick embryo scores. The eye irritation of the test subjects were classified according to the ES values in the table. The product is subjected to self-contrast from front to back: no bleeding, coagulation and vascular dissolution.
The sum of the bleeding, clotting and vascular thawing levels of the chick embryos of the test used was calculated as the mean ES score according to the following formula:
detection result: the eye cream prepared in example 1 of the present application was subjected to end point scoring according to the end point scoring criteria shown in the following table 4, and as shown in fig. 5, under the experimental conditions, the eye cream sample prepared in example 1 has an ES score of 0, an end point score ES of no more than 12, and the tested sample is non/slightly irritating and meets the SN/T2329-2009 criterion.
Table 4 endpoint scoring criteria
Endpoint scoring Irritation classification
ES≤12 No/light irritation
12＜ES≤16 Moderate irritation
ES≥16 Strong irritation/corrosiveness
(5) And (3) detecting anti-aging performance: (evaluation of anti-aging ability of eye cream Using elastase content)
A. Test materials: the device comprises an enzyme label instrument, a water-proof constant temperature incubator, a high-speed table refrigerated centrifuge, a pipette 20-200uL and a pipette 100-1000uL;
B. sample processing and related reagents:
the eye cream sample prepared in example 1 was diluted with PBS to a test solution having a concentration of 0.015625%, 0.0078125%, 0.00390625%, 0.001953125% of the original sample;
0.04mg/mL elastase solution, 0.065mg/mL N-succinyl-Ala-Ala-Ala-p-nitroaniline solution, 40ug/mL Sivelalase solution;
C. the detection process comprises the following steps:
1. preparing a sample tube: taking 60uL of sample solution and 60uL of elastase solution, slightly shaking the sample solution and the elastase solution in a test tube, standing the sample solution at 37 ℃ for 5 minutes, adding 60uL of substrate solution, and shaking the sample solution and the elastase solution;
2. sample background preparation: taking 60uL of sample solution and 60uL of elastase solution, slightly shaking the sample solution and the elastase solution in a test tube, standing the sample solution at 37 ℃ for 5 minutes, adding 60uL of 10% DMSO solution, and shaking the sample solution and the elastase solution;
3. preparing an enzyme tube: taking 60uL of solvent and 60uL of elastase liquid, slightly shaking the solvent and the elastase liquid uniformly in a test tube, standing the mixture at 37 ℃ for 5 minutes, adding 60uL of substrate solution, and shaking the mixture uniformly;
4. preparing enzyme tube background: taking 60uL of solvent and 60uL of elastase liquid, slightly shaking the solvents in a test tube, standing the solvents at 37 ℃ for 5 minutes, adding 60uL of 10% DMSO solution, and shaking the solvents; each reaction solution was reacted at 37℃for 30 minutes, 180uL was transferred to a 96-well ELISA plate, and absorbance at 405nm was measured.
Detection result:
as shown in figure 2, the inhibition rate of the prepared eye cream with four non-toxic concentrations on elastase is above 90%, namely the eye cream prepared in the embodiment 1 of the application has an inhibition effect on elastase, namely the eye cream has an obvious anti-aging effect.
(6) And (3) detecting oxidation resistance: (pair H) 2 0 2 Detection of the protective action of induced cellular oxidative stress
Culturing HaCaT cells:
HaCaT cells are respectively cultured in DMEM medium containing 10% fetal calf serum and 1% diabody, and placed in a medium containing 5% CO by volume 2 Culturing in a cell incubator at 37 ℃ until the cell reaches 80% -90% fusion degree according to the growth condition of the cell, carrying out digestion and passage by using-0.25% trypsin, and then taking the cell in the logarithmic phase for experiment;
B.H 2 0 2 determination of the damage model:
selection of HaCaT cells in logarithmic growth phase to give 1×10 5 cell suspensions of cells/mL were seeded at 100. Mu.L per well in 96-well plates with 6 duplicate wells per group and placed in a volume fraction of 5% CO 2 Culturing in a cell culture incubator at 37deg.C for 24 hr, and adding H with final volume concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7mmol/L 2 O 2 Incubation was continued for 24h.
Cell viability was measured by MTT method and H was selected with cell viability approaching 80% 2 O 2 The concentration was taken as the optimum acting concentration. Finally 500uM is selected as a subsequent experiment H 2 0 2 Concentration;
C. toxicity of eye cream samples prepared in example 1 on HaCaT cells:
HaCaT cells were grown according to 1X 10 5 cells/mL were inoculated in 96-well plates, 100. Mu.L per well, 6 duplicate wells were placed per group, and after 24h incubation in cell culture chambers,cells were treated with medium (8 concentration gradients of 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.0078125%, 0.00390625%) containing different concentrations of the resulting ocular samples prepared in example 1, and after further incubation for 24 hours, the safe dose of product to cells was determined by MTT method. Three concentrations of approximately 90% cell viability were finally selected for subsequent experiments: 0.00390625%, 0.0078125%, 0.015625%;
D. eye cream sample pair H prepared in example 1 2 0 2 Protection of induced HaCaT cell viability:
taking HaCaT cells in logarithmic growth phase, digesting with pancreatin and preparing single cell suspension with cell density of 1×10 5 cells/mL were seeded in 96-well plates at 100. Mu.L per well and placed in a solution containing 5% CO by volume 2 Culturing in a cell culture incubator at 37deg.C, adding 100ul of product (0.00390625%, 0.0078125%, 0.015625%) into the pre-protection group after cell adhesion, incubating for 24 hr, and pre-protecting group and H 2 O 2 The injury groups respectively use H-containing 2 0 2 HaCaT cells were treated with medium (final volume 500. Mu.M) for 24h, whereas the blank was added with an equal volume of medium, 6 duplicate wells were placed in each group, and after incubation for 24h, cell viability was determined by MTT method.
The detection results are shown in fig. 3: haCaT cells were isolated from H 2 O 2 After the treatment and molding, the cell activity is reduced; compared with the model group, the eye cream prepared in the embodiment 1 can improve the cell activity of HaCaT cells after injury, namely the application can reduce H 2 O 2 The anti-oxidation protective effect is exerted on the damage of cells and the increase of the survival quantity of the cells, and the anti-oxidation protective effect is effective.
(7) Detection of protection of UVB-induced photoaged cells:
culturing HaCaT cells:
HaCaT cells are respectively cultured in DMEM medium containing 10% fetal calf serum and 1% diabody, and placed in a medium containing 5% CO by volume 2 Culturing in a cell culture incubator at 37deg.C, according to the growth condition of cellsWhen the cells reach 80% -90% of fusion degree, digestion and passage are carried out by using 0.25% of trypsin, and then the cells in the logarithmic growth phase are taken for experiment;
determination of UVB photodamage model:
selection of HaCaT cells in logarithmic growth phase to give 1×10 5 cell suspensions of cells/mL were seeded at 100. Mu.L per well in 96-well plates with 6 duplicate wells per group and placed in a chamber containing 5% CO by volume 2 Culturing in a cell incubator at 37deg.C for 24 hr, removing culture solution after cell state is stable, washing twice with PBS, and respectively using irradiation dose of 0mj/cm 2 、30mj/cm 2 、60mj/cm 2 、90mj/cm 2 、120mj/cm 2 、150mj/cm 2 The UVB light source of (2) damages cells, and the cells are continuously incubated for 24 hours;
finally, detecting the cell survival rate by using an MTT method, selecting a UVB dose with the cell survival rate close to 80% as an optimal acting dose, and finally selecting 60mj/cm with smaller cell damage 2 (survival about 80%) as UVB exposure dose for subsequent experiments;
C. protection of UVB-induced HaCaT cell viability by eye cream prepared in example 1:
taking HaCaT cells in logarithmic growth phase, digesting with pancreatin and preparing single cell suspension with cell density of 1×10 5 cells/mL were seeded in 96-well plates at 100. Mu.L per well and placed in a solution containing 5% CO by volume 2 Culturing in a 37 ℃ cell incubator, adding 100ul of product (0.00390625%, 0.0078125%, 0.015625%) into the pre-protection group after cell adhesion, incubating for 24h, discarding old culture solution, washing twice with PBS, covering the control group with tinfoil, and applying doses of 60mj/cm to the pre-protection group and UVB injury group respectively 2 The HaCaT cells were irradiated with UVB, PBS was discarded, 100ul of complete medium was added to each well, incubation was continued for 24h, and cell viability was determined by MTT method.
The detection results are shown in fig. 4: compared with a blank control, the cell viability of the UVB irradiation group is reduced, and compared with a product treatment group irradiated by the UVB, the cell viability can be obviously improved, and the three concentrations are higher than those of the UVB irradiation group, namely the eye cream prepared in the example 1, and the eye cream has the effect of resisting the UVB to induce the apoptosis of HaCaT cells.
The application provides an eye cream containing pomegranate flower extract and a preparation method thereof, wherein the eye cream consists of a composite plant extract, an antibacterial agent, an antioxidant, a functional auxiliary agent and deionized water, wherein the composite plant extract consists of a pomegranate flower extract, a jojoba seed extract, a European plum extract, a avocado fruit extract and a lavender flower extract, and the eye cream is added with the natural plant extract such as the pomegranate flower extract and the like to replace the traditional anti-aging component.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.

Claims (10)

1. An eye cream containing pomegranate flower extract is characterized by comprising the following components in percentage by mass:
the composite plant extract comprises the following components in percentage by mass:
2. the eye cream of claim 1, wherein the pomegranate flower extract is PLUMP Oleoactif.
3. The eye cream containing the pomegranate flower extract according to claim 1, wherein the antibacterial agent comprises the following components in percentage by mass:
0.05-0.6% of 1, 2-hexanediol;
0.15-0.9% of p-hydroxyacetophenone;
0.21-2% of lavender flower extract.
4. The eye cream containing the pomegranate flower extract according to claim 1, wherein the antioxidant comprises the following components in total mass fraction:
0.5-1.5% of hydroxy pinacolin retinol;
0.5-1.5% of tocopherol;
0.5-3% of hydroxypropyl tetrahydropyran triol.
5. The eye cream containing the pomegranate flower extract according to claim 1, wherein the humectant comprises the following components in percentage by mass:
2-5% of squalane;
0.5-2% of dimethyl silicone oil;
avocado oil 0.5-2%.
6. The eye cream of claim 1, wherein the avocado fruit extract further comprises a 10-15% by volume solution of 1, 3-propanediol.
7. The eye cream containing pomegranate flower extract according to claim 1, wherein the softener is caprylic/capric triglyceride, the skin feel modifier is panthenol, and the pH modifier is triethanolamine.
8. A method for preparing an eye cream containing a pomegranate flower extract according to any one of claims 1 to 7, comprising the steps of:
step 101, sequentially adding cetostearyl alcohol and carbomer into a stirring kettle containing glycerol, and dispersing for 3-10min to completely dissolve under the condition that the stirring temperature is 60-80 ℃ and 800-1200rpm to obtain a pre-dispersion system;
102, sequentially adding 1, 2-propylene glycol, skin feel regulator, sodium hyaluronate and partial deionized water into the pre-dispersion system in the step 101, and stirring at a temperature of
Heat-preserving and stirring for 10-20min at 80-90 ℃ and 1500-2200rpm to obtain phase A;
step 103, sequentially adding the composite plant extract, the emulsifier, the rest deionized water, the antioxidant, the softener, the beeswax, the humectant and the antibacterial agent into a stirring kettle, and stirring for 5-10min at 60-80 ℃ and 1200-1800rpm to obtain a phase B;
and 104, sequentially adding the pH regulator, the phase A in the step 102, the phase B in the step 103 and the acetyl hexapeptide-8 into a stirring kettle, and stirring uniformly to obtain the eye cream.
9. The method for preparing eye cream containing flos Granati extract according to claim 8, wherein the stirring parameter of step 104 is at 80-90deg.C,
homogenizing at 8000-8500rpm for 3-10min, cooling to 35-40deg.C, adding acetyl hexapeptide-8, stirring at 1200-1500rpm
8-15min to obtain eye cream.
10. The method of claim 8, wherein in step 102, the deionized water is 30-50% deionized water by volume.
CN202311106610.5A 2023-08-30 2023-08-30 Eye cream containing pomegranate flower extract and preparation method thereof Pending CN117137837A (en)

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Application Number Priority Date Filing Date Title
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