Preparation and application of a nourishing and anti-wrinkle facial cream

Abstract

The present invention relates to the technical field of cosmetics, and in particular to the preparation and application of a nourishing and anti-wrinkle facial cream. The facial cream comprises active ingredients such as a Dendrobium officinale extract, a Dendrobium officinale yeast fermentation filtrate, an Opuntia radiata stem extract, panthenol, an Amygdalus dulcis seed extract, β-glucan, sodium hyaluronate, and carnosine. The facial cream of the present invention has excellent nourishing and anti-wrinkle properties; and a human patch test shows no obvious reddening, and is mild and non-irritating.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to the preparation and application of a nourishing and anti-wrinkle facial cream. Background Art

[0002] As we age, the skin's ability to shrink becomes lower and lower, which leads to dull and dull facial skin, wrinkles, etc. Face cream is a relatively thick paste-like skin care product that has a good moisturizing effect and is especially suitable for use in the dry autumn and winter seasons.

[0003] As people pursue beauty, they will use cosmetics for a long time. However, the long-term use of cosmetics will make the skin more sensitive due to various factors, such as cosmetics quality, changes in skin health or environmental factors. This has led to higher and higher requirements for cosmetics to soothe and reduce skin irritation. At the same time, anti-aging is also an important performance evaluation of cosmetics by users. Therefore, it is urgent to provide a nourishing and anti-wrinkle cream to solve the problem of providing the most basic anti-aging performance and further giving the cream soothing and anti-irritation properties.

[0004] The object of the present invention is to provide a nourishing and anti-wrinkle facial cream to solve the problem that the existing facial cream needs to provide the most basic anti-aging performance and also needs to be endowed with anti-irritation performance. Summary of the invention

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides a nourishing and anti-wrinkle cream, which comprises a plant extract composition, a nourishing composition and a moisturizer.

[0006] The plant extract composition comprises Dendrobium officinale stem extract, Dendrobium officinale fermentation filtrate, Opuntia fasciata stem extract, Prunus amygdalus dulcis seed extract and shea butter.

[0007] The nourishing composition contains panthenol, beta-glucan, and carnosine.

[0008] The moisturizing agent is selected from at least any one of sodium alginate, glycerin, hyaluronic acid and betaine.

[0009] Dendrobium officinale Kimura et Migo, a perennial epiphytic herb of the genus Dendrobium in the Orchidaceae family, is distributed throughout China. It contains rich bioactive substances, mainly including dendrobium polysaccharides, alkaloids, phenanthrene compounds and amino acids. These substances have pharmacological activities such as enhancing immunity, anti-tumor and immunomodulation. After fermentation with Chinese red yeast and strange yeast, the phenanthrene and alkaloid compounds in Dendrobium officinale can be more effectively resolved, and their biological activity is stronger.

[0010] Opuntia ficus-indica, a plant of the genus Opuntia in the Cactaceae family, is native to America. Its stem contains a variety of bioactive substances, including flavonoids, saponins, polysaccharides, etc. These ingredients have a variety of pharmacological effects, such as promoting skin growth, healing wounds, anti-inflammation, anti-tumor, etc. Therefore, it is widely used in soothing, moisturizing, and repairing products.

[0011] Sweet almond (Amygdalus communis L.), a plant of the peach family, is widely distributed along the Mediterranean coast, Asia and the temperate regions of America. Its seeds contain a variety of bioactive substances, mainly including protein, fat, fiber, vitamins and minerals. In particular, it contains high levels of unsaturated fatty acids (such as oleic acid and linoleic acid). These ingredients have liver protection, anti-oxidation, anti-inflammatory and cardiovascular protection effects. They are widely used in soothing, moisturizing and repairing products.

[0012] Panthenol, also known as panthenol or provitamin B5, is a colorless to slightly yellow transparent viscous liquid with a special smell. It is a precursor of vitamin B5 (pantothenic acid) and can be converted into pantothenic acid in the body, and then participate in the metabolism of human protein, fat and sugar. It can promote wound healing because it can stimulate cell proliferation and repair the skin by converting into pantothenic acid. Secondly, panthenol also has a strong moisturizing effect, which can help improve the skin's ability to retain water, making the skin softer, smoother and more elastic. In addition, panthenol can also improve the skin's barrier function and enhance the skin's resistance to external environmental stimuli, thereby reducing the occurrence of skin sensitivity and inflammation.

[0013] β-Glucan (Beta-Glucan), also known as "immune gold", is a natural product composed of glucose molecules connected by β-1,3-bonds and β-1,6-bonds. It is widely found in plants, fungi and microorganisms, such as mushrooms, yeast, algae, etc. β-Glucan has many important biological activities and pharmacological effects, and is widely used in medicine, health products, food and other fields. In the immune system, β-Glucan plays an important role. It can enhance the activity of immune cells, promote the proliferation and differentiation of immune cells, and improve the ability of immune cells to recognize and eliminate pathogens. In addition, β-Glucan also has anti-inflammatory, anti-tumor, antioxidant and other effects, which can regulate the function of the immune system and maintain immune balance. In addition to immunomodulatory effects, β-Glucan also has many other biological activities. For example, it can enhance antioxidant capacity, scavenge free radicals, and reduce oxidative damage. In addition, β-Glucan also has antibacterial, antiviral, anti-aging, and lipid-lowering effects, which help improve human health.

[0014] Carnosine (L-Carnosine) is a dipeptide composed of β-alanine and L-histidine, which has multiple biological activities. Carnosine is present in high levels in muscle and brain tissues, and has multiple effects such as antioxidant, anti-inflammatory, anti-glycation, and chelation. Antioxidant effect: Carnosine is an effective antioxidant that can remove reactive oxygen free radicals (ROS) and α-β unsaturated aldehydes produced during oxidative stress, thereby protecting cells from oxidative damage. Anti-inflammatory effect: Carnosine has anti-inflammatory effects and can inhibit the occurrence and development of inflammatory reactions, thereby reducing the damage of inflammation to tissues. Anti-glycation effect: Carnosine can inhibit the occurrence of glycation reactions, thereby reducing the formation of AGEs (advanced glycation end products), which helps prevent the occurrence of chronic diseases such as diabetes. Chelation: Carnosine can also bind to metal ions to form stable chelates, thereby regulating the concentration and activity of metal ions in the body. In addition, carnosine also has the advantages of good water solubility, stable properties, small molecular weight, and easy absorption and utilization by the human body. Therefore, carnosine has been widely used in the fields of food additives, cosmetic additives, etc. Experiments have proved that the facial cream of the present invention has excellent nourishing and anti-wrinkle properties; among them, the combination of Dendrobium officinale extract, Dendrobium officinale yeast fermentation filtrate, Xiantao cactus stem extract, sweet almond seed extract, and shea butter has excellent effects in giving the facial cream anti-aging effects, and the combination of β-glucan and sodium hyaluronate has a significant effect in giving the facial cream nourishing effects.

[0015] Preferably, the raw materials of the plant extract composition include, by mass fraction, 0.5-3 parts of Dendrobium officinale stem extract, 0.05-1 parts of Dendrobium officinale fermentation filtrate, 0.1-1 parts of Xiantao cactus stem extract, 0.1-1 parts of sweet almond seed extract, and 0.1-1 parts of shea butter; by mass fraction ratio, the ratio of Dendrobium officinale stem extract, Dendrobium officinale fermentation filtrate, Xiantao cactus stem extract, sweet almond seed extract and shea butter is 10-30:1:0.1-20:0.1-20:0.1-20.

[0016] Preferably, the raw materials of the nourishing composition include, by mass fraction, 0.8-1 part of panthenol, 0.05-0.08 part of β-glucan, and 0.1-0.3 part of carnosine; by mass fraction ratio, the ratio of panthenol, β-glucan, and carnosine is 1:0.05-0.08:0.1-0.3.

[0017] Preferably, the moisturizing agent is at least one of trehalose, glycerin, sodium hyaluronate, betaine, and cetearyl olivate.

[0018] Preferably, the antioxidant is at least one of sodium citrate, beeswax and tocopherol acetate.

[0019] The invention provides a method for preparing the Dendrobium officinale fermentation filtrate, which comprises crushing the Dendrobium officinale stems and using fermentation bacteria to mix and ferment to obtain the Dendrobium officinale fermentation filtrate.

[0020] Preferably, the method for preparing the Dendrobium officinale fermentation filtrate is:

[0021] S1. Dry and crush the broken Dendrobium officinale, add the culture medium for sterilization, and the solid-liquid ratio is 1:5-1:15;

[0022] S2, adding fermentation bacteria to the product of S1, fermenting at a specific oxygen content, specific fermentation temperature, and specific time;

[0023] S3, collecting the filtrate of the S2 product;

[0024] S4, drying the product of S3 to prepare the fermented extract of Dendrobium officinale.

[0025] Preferably, the material-liquid ratio in S1 is 1:3.

[0026] Preferably, the specific oxygen content in S2 is 8%-12%, the specific fermentation temperature is 28-32° C., and the specific fermentation time is 24-72 hours.

[0027] Preferably, the specific oxygen content in S2 is 11%, the specific fermentation temperature is 30° C., and the specific fermentation time is 68 h.

[0028] Preferably, the fermentation bacteria in S2 are any one or both of Chinese red yeast and strange yeast.

[0029] Preferably, the fermentation bacteria in S2 are Chinese red yeast and strange yeast, and the mass ratio thereof is 1:0.1-2.

[0030] Preferably, the present invention provides a method for preparing a nourishing and anti-wrinkle facial cream, characterized in that it comprises 8-10 parts of a plant extract composition, 0.5-2 parts of a nourishing composition, 4-18 parts of a moisturizer, and 2-4 parts of an antioxidant.

[0031] Most preferably, the mass ratio of the plant extract composition, the nourishing composition, the moisturizing agent and the antioxidant is 5:1:9.5:2.

[0032] The present invention also provides a method for preparing a nourishing and anti-wrinkle cream, characterized in that the preparation method is as follows:

[0033] S1, adding water and moisturizer to obtain a mixed solution A;

[0034] S2, then add the nourishing composition and continue stirring to obtain a mixture B;

[0035] S3, then add plant extracts and antioxidants, and continue stirring to obtain the nourishing and anti-wrinkle cream.

[0036] Compared with the prior art, the invention has the following beneficial effects: the invention innovatively uses a mixture of Saccharomyces cerevisiae and Chinese red yeast to ferment Dendrobium candidum, and after fermentation, the activity and richness of phenanthrene and alkaloid substances in Dendrobium candidum are significantly improved, wherein the functionality and application pathways of natural products such as glycosides, terpenes and plant polysaccharides are significantly improved, and there are also new active ingredients such as ergothioneine and carotene that were not originally in the Dendrobium candidum extract. By combining phenanthrene and alkaloid substances in the Dendrobium candidum fermentation extract with active products such as Dendrobium candidum stem extract, Xiantao cactus stem extract, sweet almond seed extract and shea butter, and then supplemented with a nourishing composition of panthenol, β-glucan and carnosine, the prepared cream has significant nourishing, anti-wrinkle, moisturizing, soothing and other effects. It can also significantly help wound healing, promote cell migration rate, and improve the skin's own barrier repair function.

[0037] It has been found through experiments that the nourishing and anti-wrinkle cream can repair skin damage caused by various reasons, has nourishing, anti-wrinkle, moisturizing and soothing effects, and is suitable for various skin types. The present invention finds that under the premise of the same usage amount, the specific proportions of Dendrobium officinale extract, Dendrobium officinale yeast fermentation filtrate, Opuntia cactus stem extract, panthenol, sweet almond seed extract, β-glucan, sodium hyaluronate, and carnosine defined in the present invention are better than using them alone, or any three or more of them together, producing a synergistic effect, so that the nourishing, anti-wrinkle, moisturizing, and soothing effects are better. The present invention also provides the preferred formula and preparation method of the nourishing and anti-wrinkle cream. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Example 9-16 HCEC survival rate.

[0039] Figure 2 Hyaluronidase inhibition rate of Examples 9-16 and Comparative Examples 13-26.

[0040] Figure 3 TNF-α inflammatory factor inhibition rate of Examples 9-16 and Comparative Examples 13-26.

[0041] Figure 4 The inhibition rate of zebrafish tail area reduction in Examples 9-16 and Comparative Examples 13-26.

[0042] Figure 5 Hacat cell migration rate of Examples 9-16 and Comparative Examples 13-26.

[0043] Figure 6Relative transepidermal water loss rates of Examples 17-19 and Comparative Examples 27-28. DETAILED DESCRIPTION

[0044] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention is further described by the following examples. It should be understood that the examples of the present invention are only used to illustrate the technical effects of the present invention, but not to limit the scope of protection of the present invention. In the examples, the methods used are conventional methods unless otherwise specified.

[0045] Some of the raw materials in the present invention come from the following sources:

[0046] Dendrobium officinale stem extract: purchased from Xi'an Qiancao Biotechnology Co., Ltd.

[0047] Opuntia cactus stem extract: purchased from Xi'an Qiancao Biotechnology Co., Ltd.

[0048] Cactus stem extract: purchased from Xi'an Qiancao Biotechnology Co., Ltd.

[0049] Prunus amygdalus seed extract: purchased from Xi'an Qiancao Biotechnology Co., Ltd.

[0050] Shea butter: purchased from Xi'an Qiancao Biotechnology Co., Ltd.

[0051] Dendrobium officinale: purchased from Xi'an Qiancao Biotechnology Co., Ltd.

[0052] Carnosine: purchased from Nanjing Peptide Valley Biotechnology Co., Ltd.

[0053] β-Glucan: Suzhou Yaxu Fine Chemical Co., Ltd.

[0054] Sodium hyaluronate: Aivitao (Shanghai) Pharmaceutical Technology Co., Ltd.

[0055] Saccharomyces paradoxus: purchased from Ningbo Testo Biotechnology Co., Ltd.; 3000 CFU / mL.

[0056] Chinese red yeast (Rhodosporidium Toruloides): China Microorganism Culture Collection Center, No. CGMCC27505; 3000 CFU / mL.

[0057] Culture medium: self-prepared.

[0058] The preparation method of the Dendrobium officinale fermentation filtrate of the present invention is as follows:

[0059] S1. Dry and crush the broken Dendrobium officinale, add the culture medium for sterilization, and the solid-liquid ratio is 1:1-3;

[0060] S2, adding fermentation bacteria to the product of S1, fermenting at a specific oxygen content, specific fermentation temperature, and specific time;

[0061] S3, collecting the filtrate of the S2 product;

[0062] S4, drying the product of S3 to prepare the fermented extract of Dendrobium officinale.

[0063] The specific steps of the medium configuration method described in S1 are as follows:

[0064] 1. Dissolve 10g Yeast Extract and 20g Peptone in 900ml water. If making plates, add 20g agar powder.

[0065] 2. High pressure 115 degrees for 15 minutes;

[0066] 3. Add 100ml20gDextrose (glucose) (glucose solution is added after sterilization);

[0067] Note: Glucose, yeast extract, and eptone solutions may react chemically at high temperatures, causing changes in the composition of the culture medium, so they must be sterilized separately before mixing. Glucose can be sterilized by filtration or sterilized at 115℃ for 15 minutes.

[0068] The specific parameters are shown in Table 1.

[0069] Table 1 Fermentation process parameters of Dendrobium officinale

[0070] step Strange yeast Chinese red yeast Oxygen content Fermentation temperature Fermentation time Material-liquid ratio Example 1 1wt% 0.1wt% 8％ 28℃ 24h 1：1 Example 2 1wt% 1wt% 11％ 30℃ 68h 1：3 Example 3 1wt% 2wt% 12％ 32℃ 72h 1；5 Comparative Example 1 0wt% 1wt% 11％ 30℃ 68h 1：3 Comparative Example 2 1wt% 0wt% 11％ 30℃ 68h 1：3

[0071] The preparation of the plant extract composition comprises the following steps:

[0072] S1 Weigh a specific mass portion of plant extract in a specific ratio and place it in a beaker.

[0073] The specific parameters are shown in Table 2.

[0074] Table 2 Composition ratio

[0075]

[0076]

[0077] Note: The pretreatment methods of the root, stem and leaf samples of Dendrobium officinale are the same.

[0078] A preparation method of a facial cream comprises the following steps:

[0079] S1, adding water and moisturizer to obtain a mixed solution A;

[0080] S2, adding the nourishing composition to A and continuing to stir to obtain a mixture B;

[0081] S3, add the plant extract to B, continue stirring, and obtain the nourishing and anti-wrinkle cream. Specific parameters are shown in Table 3-4.

[0082] Table 3 Preparation mass fraction parameters of active ingredients in facial cream

[0083]

[0084]

[0085] Table 4 Addition ratio of each component of cream

[0086] Experimental Group Active ingredients Active ingredient number water Embodiment 17 30wt% Embodiment 11 70wt% Embodiment 18 40wt% Embodiment 11 60wt% Embodiment 19 25wt% Embodiment 11 75wt% Comparative Example 27 0wt% Embodiment 11 100wt% Comparative Example 28 50wt% Embodiment 11 50wt%

[0087] 1. Safety Testing

[0088] With reference to the 2015 Technical Specifications for Safety of Cosmetics, the compositions of Examples 9 to 16 were evaluated for cosmetic irritation. The test method was a skin patch test, and 80 randomly distributed people aged 16 to 65 were tested.

[0089] Test method: Put the test substance into the patch tester, the dosage is 0.020-0.025g, cover the patch tester with the test substance on the back or forearm of the subject with non-irritating cloth-based tape, and gently press it with the palm to make it evenly adhere to the skin surface for 24 hours. After removing the test patch tester, observe the skin reaction after the indentation disappears 30 minutes later. If the result is negative, observe again 24 hours and 48 hours after the patch test.

[0090] Evaluation criteria:

[0091] Grade 0: negative reaction;

[0092] Grade 1: Suspicious reaction, only slight erythema;

[0093] Grade 2: weak positive reaction, erythema, infiltration, edema, and papules may be present;

[0094] Grade 3: Strong positive reaction, erythema, infiltration, edema, papules, and the reaction may extend beyond the test area;

[0095] Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent herpes, and the reaction exceeds the test area.

[0096] Test results: Skin reactions were negative in all subjects.

[0097] The above test results show that the composition provided by the present invention is mild and non-irritating to the skin and is safe to use.

[0098] 2. Cytotoxicity Test

[0099] Take human corneal epithelial cells HCEC with stable growth state and adjust the cell concentration to 1×10 5 / mL, 100μL per well. Inoculate into 96-well cell culture plates (except the outermost wells and blank control wells). Add 200μL of PBS to the outermost wells of the cell culture plate to reduce the evaporation of the culture medium. Place in a carbon dioxide incubator and culture at 37℃±1℃, CO2 concentration 5.0%±1% until the cell fusion reaches more than 90%. Add samples, the sample concentration is 10mg / L, set up blank control and sample group, 100μL per well. Start adding samples (sample group) and PBS (blank control) at the same time interval between each two columns to ensure that the intervention time of each test substance on the cells is 5min. After 5min, remove the test substances in turn, add PBS to each well to wash the test substances, and wash twice. When using a vacuum suction pump or a discharge gun to remove the liquid in the well, be careful to avoid sucking away the surviving cells and affecting the experimental results. Add 100μL of complete culture medium and 10μL of CCK-8 solution to each well, and place in an incubator for about 2h. At a wavelength of 450 nm, the absorbance (OD) value was measured and the cell viability was calculated; the results are shown in Table 4.

[0100] Formula (1)

[0101] Table 5 Cytotoxicity test

[0102]

[0103]

[0104] According to the experimental results in Table 5, the cell survival rate was maintained above 95%. The combination of the present experiment had little effect on the viability of HCEC cells.

[0105] 3. Hyaluronidase Activity Inhibition Experiment

[0106] Hyaluronic acid, also known as hyaluronic acid, is a powerful moisturizer. It can attract and lock in moisture, maintain skin moisture, and improve dry, rough and dehydrated skin conditions. Hyaluronic acid can also enhance the biological activity of cells and has a strong synergistic effect on repairing and protecting the skin. Therefore, by inhibiting hyaluronidase, the decomposition rate of hyaluronic acid in the skin is reduced, thereby achieving long-term moisture retention and repair of the skin, which is of great significance.

[0107] (1) Take four groups of clean test tubes, label them A, B, C, and D, add 50 μL of the sample (10 mg / L) to test tubes A and B, add 50 μL of distilled water to test tubes C and D, add 50 μL of 500 U / mL hyaluronidase solution to test tubes A and C, add 50 μL of pH 5.6 acetate buffer to test tubes B and D, and place the test tubes in a 37°C incubator for 20 min.

[0108] (2) Add 10 μL of 2.5 mol / L CaCl2 solution to each of the four test tubes and place them in an incubator at 37°C for 20 min.

[0109] (3) Add 50 μL of 0.5 mg / mL sodium hyaluronate solution to test tubes A and C, and add 50 μL of pH 5.6 acetate buffer to test tubes B and D. Place the test tubes in a 37°C incubator for 40 min and then take them out and place them at room temperature for 10 min.

[0110] (4) Add 50 μL of distilled water, 10 μL of 5 mol / L NaOH solution, and 50 μL of acetylacetone solution to the four test tubes respectively. Place the test tubes in a boiling water bath for 30 min, then in an ice bath for 10 min, and finally at room temperature for 10 min.

[0111] (5) Add 100 μL of P-DAB reagent to each test tube, and then measure the absorbance at 530 nm using an ELISA reader.

[0112] Formula (2)

[0113] Wherein: A is the OD value of the sample solution; B is the OD value of the sample blank; C is the OD value of the control sample solution; D is the OD value of the control sample blank.

[0114] Table 6 Hyaluronidase activity inhibition rate

[0115]

[0116]

[0117] From the analysis of the results in Table 6: It can be seen from the results of Examples 9-16 that the compositions provided in the embodiments of the present invention have good hyaluronidase inhibitory effects, especially Example 11 has a better effect, indicating that using Example 2 as the fermentation process parameters of Dendrobium officinale can effectively improve the inhibitory effect of the cream on hyaluronidase.

[0118] From the results of Example 11 and Examples 13-16, within the mass range of the components provided by the present invention, the cream has a significant effect of inhibiting hyaluronidase. The effect of Example 11, that is, when the mass fraction ratio is 5:1:9.5:2, the effect of inhibiting hyaluronidase is the best.

[0119] Comparing Example 11 with Comparative Examples 13-16, Example 11 has the best effect, indicating that within the component mass range provided by the present invention, the cream has a significant effect of inhibiting hyaluronidase, and excessive addition cannot significantly improve its hyaluronic acid inhibition effect.

[0120] Comparative Example 11 and Comparative Examples 17-21 show that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved without any one of them.

[0121] Comparative Example 11 and Comparative Examples 22-24 show that using the extraction site defined by the present invention can enable the present invention to achieve maximum efficacy in inhibiting hyaluronic acid, and changing any extraction site not defined by the present invention cannot achieve the effect achieved by the present invention.

[0122] By comparing the results of Example 11 with Comparative Examples 25-26, it can be seen that the use of the fermentation process defined in the present invention can enable the present invention to achieve the maximum efficacy in inhibiting hyaluronic acid, and changing any fermentation process not defined in the present invention cannot achieve the effect achieved by the present invention.

[0123] IV. Inflammatory factor inhibition test

[0124] TNF-α is the most common inflammatory factor in inflammatory reactions. It can induce the phenotype of vascular endothelial cells to change to an inflammatory type, leading to changes in the body's metabolism and hemodynamics, and promoting the production of inflammatory mediators. Inflammatory mediators can increase vascular permeability, causing local congestion and edema, and may stimulate nerve endings to cause itching and pain. Therefore, the inhibition of TNF-α helps soothe and repair the skin.

[0125] RAW264.7 cells grown to the logarithmic phase were cultured at 1.5×10 5 Cells were inoculated at a density of 100 μg / mL in a 24-well plate, with 800 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to near fusion, 50 μL of the sample concentration of 10 mg / L was administered, with 3 replicates per group. The positive control was 50 μL of 10 μg / mL dexamethasone, and 1 μg / mL lipopolysaccharide (LPS) was used for stimulation 1 hour later. After incubation in a cell culture incubator for 24 hours, the cell culture fluid was transferred to a centrifuge tube, centrifuged at 5000 r / min for 10 minutes, and the cell supernatant was collected and tested according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.

[0126] Formula (3)

[0127] Table 7 Results of RAW secretion of TNF-α

[0128]

[0129]

[0130] From the results analysis of Table 7: From the results of Examples 9-16, it can be seen that the composition provided by the embodiments of the present invention has good TNF-α inhibitory effect, especially Example 11 has a better effect, which means that using Example 2 as the fermentation process parameter of Dendrobium officinale can effectively improve the inhibitory effect of the cream on TNF-α.

[0131] From the results of Example 11 and Examples 13-16, within the mass range of the components provided by the present invention, the cream has a significant effect of inhibiting TNF-α. The effect of Example 11, i.e., when the mass fraction ratio is 5:1:9.5:2, the TNF-α inhibition effect is the best.

[0132] Comparing Example 11 with Comparative Examples 13-16, Example 11 has the best effect, indicating that within the mass range of the components provided by the present invention, the cream has a significant effect of inhibiting TNF-α, and excessive addition cannot significantly improve its TNF-α inhibitory effect.

[0133] Comparative Example 11 and Comparative Examples 17-21 show that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved without any one of them.

[0134] Comparative Example 11 and Comparative Examples 22-24 show that using the extraction site defined by the present invention can enable the present invention to achieve maximum efficacy in inhibiting TNF-α, and changing any extraction site not defined by the present invention cannot achieve the effect achieved by the present invention.

[0135] By comparing the results of Example 11 with Comparative Examples 25-26, it can be seen that the use of the fermentation process defined by the present invention can enable the present invention to achieve the maximum efficacy in inhibiting TNF-α, and changing any fermentation process not defined by the present invention cannot achieve the effect achieved by the present invention.

[0136] 5. Effect of the composition on moisturizing zebrafish

[0137] The zebrafish epidermis has a certain osmotic pressure tolerance range, and water loss will occur if the tolerance range is exceeded. Using a high osmotic pressure solution to treat zebrafish will cause its tail to shrink and become smaller due to water loss. Therefore, an induced model of zebrafish hydration and moisturizing can be established to evaluate the effect of moisturizing products.

[0138] (1) Breeding: Zebrafish were paired at a male-female ratio of 2:1, and the eggs were collected and incubated overnight (28°C incubator).

[0139] (2) Experimental groups: The experiment set up a blank control group and an experimental group. The experimental groups included a model group, a positive group, and a sample group, with 8 2 dpf fry in each group.

[0140] (3) Solution preparation: Accurately weigh 1 mg of sample and prepare the sample solution to be tested, use urea (0.05%) as the positive control for the experiment, and add 1 mg of sample to each well. After the administration is completed, place the 6-well plate in a 28°C incubator in the dark for 24 hours.

[0141] Formula (4)

[0142] Test results: as shown in Table 8 below.

[0143] Table 8 Zebrafish moisturizing effect test

[0144] Experimental Group Zebrafish tail area reduction inhibition rate Positive group 1 blank 0 Example 9 0.55 Example 10 0.43 Embodiment 11 0.41 Example 12 0.37 Example 13 0.44 Embodiment 14 0.46 Embodiment 15 0.42 Example 16 0.47 Comparative Example 13 0.44 Comparative Example 14 0.45 Comparative Example 15 0.54 Comparative Example 16 0.43 Comparative Example 17 0.49 Comparative Example 18 0.42 Comparative Example 19 0.47 Comparative Example 20 0.38 Comparative Example 21 0.42 Comparative Example 22 0.41 Comparative Example 23 0.32 Comparative Example 24 0.37 Comparative Example 25 0.31 Comparative Example 26 0.28

[0145] From the results analysis of Table 8: From the results of Examples 9-16, it can be seen that the composition provided by the embodiments of the present invention has a significant effect of inhibiting the reduction of the zebrafish tail area, especially Example 11 has a better effect, which means that using Example 2 as the fermentation process parameter of Dendrobium officinale can effectively improve the effect of inhibiting the reduction of the zebrafish tail area.

[0146] From the results of Example 11 and Examples 13-16, within the mass range of the components provided by the present invention, the cream has a significant effect of inhibiting the reduction of the zebrafish tail area. The effect of Example 11, i.e., when the mass fraction ratio is 5:1:9.5:2, the effect of inhibiting the reduction of the zebrafish tail area is the best.

[0147] Comparing Example 11 with Comparative Examples 13-16, Example 11 has the best effect, indicating that within the component mass range provided by the present invention, the cream has a significant effect of inhibiting the reduction of the zebrafish tail area, and excessive addition cannot significantly improve its inhibitory effect on the reduction of the zebrafish tail area.

[0148] Comparative Example 11 and Comparative Examples 17-21 show that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved without any one of them.

[0149] Comparative Example 11 and Comparative Examples 22-24 show that using the extraction site defined by the present invention can enable the present invention to achieve maximum efficacy in inhibiting the reduction of the zebrafish tail area, and changing any extraction site not defined by the present invention cannot achieve the effect achieved by the present invention.

[0150] By comparing the results of Example 11 with Comparative Examples 25-26, it can be seen that the use of the fermentation process defined by the present invention can enable the present invention to achieve the maximum effect in inhibiting the reduction of the zebrafish tail area, and changing any fermentation process not defined by the present invention cannot achieve the effect achieved by the present invention.

[0151] VI. Effects of the composition on the scratch migration of Hacat cells

[0152] The degree of cell migration has an important impact on wound healing and skin repair. In the process of wound healing, cell migration is an important step for epidermal cells to cover the wound, so the higher the cell migration rate, the faster the wound heals. At the same time, cell migration is also involved in the repair process after skin damage. If cell migration is impaired, it will lead to skin repair disorders, which in turn cause various skin diseases.

[0153] Inoculate cells into 12-well plates at the corresponding inoculation density and incubate overnight at 37°C in a CO2 incubator. When the cell plating rate in the 12-well plate reaches about 80%, scratch the sample group and the solvent control group. After the scratching, wash the cells with PBS to remove dead cells, and then add fresh EGF-free keratinocyte culture fluid, add 50μL of 10mg / L according to Examples 5, 7-9, and Comparative Examples 1-16, and take photos and record using an inverted microscope. Incubate in a 37°C, 5% CO2 incubator for 24h, take photos and record again, and use ImageJ software for statistical analysis.

[0154] Table 9 Hacat cell migration rate

[0155]

[0156]

[0157] From the results analysis of Table 9: From the results of Examples 9-16, it can be seen that the composition provided by the embodiments of the present invention has the effect of significantly improving the cell migration rate, especially the effect of Example 11 is better, indicating that using Example 2 as the fermentation process parameter of Dendrobium officinale can effectively improve the effect of cell migration rate.

[0158] From the results of Example 11 and Examples 13-16, within the mass range of the components provided by the present invention, the cream has a significant effect of improving the cell migration rate. The effect of Example 11, i.e., when the mass fraction ratio is 5:1:9.5:2, the effect of improving the cell migration rate is the best.

[0159] Comparing Example 11 with Comparative Examples 13-16, Example 11 has the best effect, which shows that within the mass range of the components provided by the present invention, the cream has the effect of significantly improving the cell migration rate, and excessive addition cannot significantly improve the cell migration rate.

[0160] Comparative Example 11 and Comparative Examples 17-21 show that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved without any one of them.

[0161] Comparative Example 11 and Comparative Examples 22-24 show that using the extraction site defined by the present invention can enable the present invention to achieve maximum efficacy in improving cell migration rate, and changing any extraction site not defined by the present invention cannot achieve the effect achieved by the present invention.

[0162] By comparing the results of Example 11 with Comparative Examples 25-26, it can be seen that the use of the fermentation process defined in the present invention can enable the present invention to achieve the maximum effect in improving cell migration rate, and changing any fermentation process not defined in the present invention cannot achieve the effect achieved by the present invention.

[0163] 7. Preparation and stability testing of skin care gel

[0164] According to the above formula design, Examples 17-19 and Comparative Examples 23-24 were prepared and tested.

[0165] 1. Heat resistance test: Adjust the constant temperature incubator to 40℃, put the sample into a transparent glass bottle with a volume of 10ml / bottle, seal it and place it in a constant temperature incubator. Take it out after three months, return it to room temperature, and observe the changes in appearance.

[0166] 2. Cold resistance test: Adjust the constant temperature incubator to -10℃, put the sample into a transparent glass bottle with a volume of 10ml / bottle, seal it and place it in a constant temperature incubator. Take it out after three months, return it to room temperature, and observe the changes in appearance.

[0167] 3. Room temperature test: Take a transparent glass bottle, fill it with 10 ml of sample per bottle, seal it and place it at room temperature for 6 months, then observe the changes in appearance.

[0168] 4. Light aging test: Put the sample into a transparent glass bottle with a volume of 10 ml / bottle. After sealing, place it under incandescent light for three months and observe the changes in appearance.

[0169] No precipitation or settling was observed in the heat resistance test, cold resistance test, room temperature test, and light aging test, and the original physical properties were maintained.

[0170] 8. Test on the immediate repair effect of skin care gel on human body

[0171] Transepidermal water loss (TEWL) is an important parameter for evaluating skin barrier function. It indicates the loss of water from the deep dermis through evaporation of the epidermis. The higher the TEWL, the more water is lost through the skin and the weaker the skin's moisturizing ability.

[0172] The transepidermal water loss rate is closely related to the hydration of the stratum corneum and is a commonly used indicator reflecting the barrier function of the stratum corneum. In the development of cosmetics, the efficacy of moisturizing cosmetics can be evaluated by testing the TEWL value of skin water loss. In addition, the transepidermal water loss rate can also be used in allergic patch tests, contact dermatitis, physical therapy, burns and new tissue monitoring to promptly detect whether the protective function of the skin has been damaged.

[0173] Therefore, transepidermal water loss rate is an important parameter for assessing skin health and is of great significance to the research, development and evaluation of moisturizing products.

[0174] 50 healthy subjects aged 18-60 years old with no injuries on the inner arm were selected and divided into groups, with 10 people in each group, including 5 males and 5 females. A sample group and a blank group (i.e., negative control group: no test sample) were set up, and the test area was randomly distributed 1 cm inside the volunteer's arm. 2 Area. The forearms (two areas) of the above-mentioned subjects were torn off with medical tape 20 times to induce damage to the skin barrier function, and then 5 mg / mL histamine was added to model for 10 minutes (obvious erythema appeared). One area was treated with 1g of the test product, and the other area was blank as a negative control area. The transepidermal water loss rate (TEWL) of the forearm skin was collected before tape tearing off (T0), before using the product (i.e., immediately after histamine) (T1), and 2 hours after using the product (T2). The difference in skin index changes between the product-treated area and the negative control area before and after using the product was used to evaluate the short-term repair effect of the product on skin barrier damage caused by tape tearing and histamine-induced skin irritation, and the average value was taken. The results are shown in Table 10 below.

[0175] Formula (5)

[0176] Table 10 Relative water loss rate of epidermis

[0177] Experimental Group Relative transepidermal water loss Embodiment 17 -2.01％ Embodiment 18 -11.29％ Embodiment 19 -2.26％ Comparative Example 27 2.53％ Comparative Example 28 -1.19％

[0178] Result analysis: From the results of Examples 17-19, it can be seen that the facial cream prepared by the present invention has significant moisturizing and repairing effects, and Example 18, i.e., when the active ingredient is 40 wt%, has the best moisturizing and repairing effects.

[0179] Comparing Example 18 with Comparative Examples 27-28, Example 18 has the best effect, which shows that the facial cream prepared by the present invention has good moisturizing and repairing effects, and too high an addition amount cannot effectively improve the moisturizing and repairing effects. Therefore, the facial cream prepared by the process, extraction site, and proportion of each component specified by the present invention can exert the maximum effect.

[0180] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A nourishing and anti-wrinkle facial cream, characterized in that: The facial cream comprises the following components in parts by weight: 8-11 parts of plant extract composition; 0.5-4 parts of nourishing composition; Moisturizer 18-21 parts; The plant extract composition contains the following components in parts by weight: 0.5-5 parts of Dendrobium officinale stem extract; 0.01-1 part of Dendrobium officinale fermentation filtrate; 0.1-2 parts of Xiantao cactus stem extract; 0.1-2 parts of sweet almond seed extract; Shea butter 0.1-2 parts; The nourishing composition comprises the following components in parts by weight: 0.5-2 parts of panthenol; β-glucan 0.05-1 part; Carnosine 0.01-1 part; The preparation method of the Dendrobium officinale fermentation filtrate is as follows: S1. Dry and crush the broken Dendrobium officinale, add the culture medium for sterilization, and the solid-liquid ratio is 1:5-1:15; S2, adding fermentation bacteria to the product of S1, fermenting at a specific oxygen content, specific fermentation temperature, and specific time; S3, collecting the filtrate of the S2 product; S4, drying the product of S3 to prepare a fermented extract of Dendrobium officinale; In S2, the fermentation strains are Chinese red yeast and strange yeast, and the mass ratio thereof is 1:0.1-2; In S2, the fermentation oxygen content is 8%-12%, the fermentation temperature is 28°C-32°C, and the fermentation time is 24h-72h.

2. The facial cream according to claim 1, characterized in that: The moisturizing agent is at least one of trehalose, glycerin, sodium hyaluronate, betaine, and cetearyl olive oil ester.

3. The facial cream according to claim 1, characterized in that: It also includes 2-5 parts of antioxidant; the antioxidant is at least one of sodium citrate, beeswax and tocopherol acetate.

4. The facial cream according to claim 1, characterized in that Its preparation method is: S1, adding water and moisturizer to obtain a mixed solution A; S2, adding the nourishing composition to the mixed solution A, and continuing to stir to obtain a mixture B; S3, adding the plant extract composition to the mixed solution B, and continuing to stir to obtain the nourishing and anti-wrinkle cream.

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