An anti-aging composition containing human albumin and its preparation method and application

Abstract

The present invention discloses an anti-aging composition containing human albumin, a preparation method and application thereof, and relates to the field of cosmetics. The anti-aging composition of the present invention comprises 5 to 20 parts by mass of human albumin, 1.0 to 5.0 parts by mass of ginseng stem and leaf extract, and 0.10 to 0.50 parts by mass of forest frog oil. Human albumin, ginseng stem and leaf extract, and forest frog oil synergize to effectively promote the growth of fibroblasts, have a significant inhibitory effect on collagenase, and have a significant anti-wrinkle effect and good anti-aging effect. The anti-aging composition of the present invention has simple ingredients and a simple preparation method, and has broad application prospects in the field of cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to an anti-aging composition containing human albumin, a preparation method thereof and an application thereof. Background Art

[0002] Human skin is an organ that easily ages. As we age, the signs of skin aging also appear: increased wrinkles, loose and yellowed skin, reduced luster, smoothness, and lustre, coarsening of the skin texture, increased pigmentation or the appearance of depigmentation, etc. It is generally believed that the causes of skin aging are mainly endogenous and exogenous factors. Endogenous factors include: (1) genetic factors, as the order of chromosome polymerization reactions caused by genetic inheritance will change with age; (2) hormone deficiency, as hormone deficiency leads to hormonal aging. For example, estrogen deficiency can reduce the function of the skin's connective tissue and lead to various skin changes. Exogenous factors include: (1) pollution, namely, the pollution caused by the ever-increasing exhaust gas and various pollutants in the atmosphere forms a large number of free radicals. The attack of free radicals leads to the death of skin cells and rapid aging, such as skin suffocation, clogged pores, and skin fragility; (2) photoaging, namely, the accumulation of UVA / UVB rays produces some very harmful effects on the skin, such as dermal tissue disorder and thinning of the epidermal dermal connective tissue that controls the fine-tuning of the skin. In addition, excessive exposure to ultraviolet rays is also a major cause of premature aging symptoms on facial skin.

[0003] Anti-aging cosmetics target the characteristics and causes of aging skin. On the basis of moisturizing and sun protection, they add active nutrients to enhance skin nutrient absorption, while further smoothing the skin to achieve the purpose of delaying aging and removing wrinkles.

[0004] With the restriction of the use of many synthetic chemicals, "green" has become the development direction of cosmetics in the 21st century. Consumer demand for safe and non-irritating cosmetics has promoted the use of natural active ingredients in cosmetics. Natural ingredients with physiological activities such as anti-free radical, antioxidant, anti-radiation, antibacterial, and anti-inflammatory properties are gaining increasing attention. Currently, the development of natural active ingredients for use in anti-aging cosmetics has become a trend.

[0005] Existing anti-aging products include albumin, but albumin primarily serves as an auxiliary functional ingredient or works in conjunction with other active peptides. For example, in a stem cell skin care composition disclosed in CN106983713A, its preparation method, and its use method, the serum albumin content is only 1% of the main active ingredient, stem cell extract. In an anti-aging composition, a lyophilized powder containing the anti-aging composition, and its preparation method disclosed in CN110613628A, serum albumin is compounded with specific oligopeptides to produce anti-aging effects, but serum albumin is primarily used as a protein protectant. Currently, there are relatively few reports on anti-aging products using serum albumin as the main active antioxidant ingredient. Summary of the Invention

[0006] In view of the problems and shortcomings of the prior art, the present invention provides an anti-aging composition containing human albumin, a preparation method and application thereof. The composition of the present invention comprises human albumin, ginseng stem and leaf extract and Rana sylvatica oil.

[0007] Human serum albumin (HSA) is a monomeric protein produced by the liver. Mature HSA contains 585 amino acid residues and has a molecular weight of 66 kDa. Albumin is rich in free sulfhydryl groups and possesses antioxidant properties, scavenging free radicals (such as hypochlorous acid (HOCl) and peroxynitrite (ONOOH)) and donating sulfhydryl groups (-SH), thereby exerting anti-aging effects.

[0008] Ginseng stems and leaves: These are the dried stems and leaves of Panax ginseng (CA Mey.), a plant of the Araliaceae family. Ginseng stems and leaves are cold in nature, with a bitter and sweet taste. They enter the lung and stomach meridians, tonifying Qi, benefiting the lungs, dispelling summer heat, and promoting salivation. Pharmacological research indicates that the ginsenosides found in ginseng stems and leaves possess antioxidant and anti-inflammatory properties, and can delay cell aging. Ginseng polysaccharides also have a positive impact on enhancing immune function and promoting hematopoiesis.

[0009] Rana oil is harvested and dried from the oviduct of the female Chinese wood frog (Rana temporariachensinensis David), a species of the Ranidae family. It has a sweet and salty flavor and a neutral nature. It enters the lung and kidney meridians, nourishing the kidneys and replenishing essence, and nourishing yin and moistening the lungs. Scientific research has shown that Rana oil is rich in bioactive peptides, which are effective in regulating collagen, elastin, and melanin synthesis in dermatology. It also possesses antioxidant, anti-fatigue, and anti-inflammatory properties.

[0010] In a first aspect, the present invention provides an anti-aging composition containing human albumin, wherein the composition comprises the following components in parts by weight: 5 to 20 parts by weight of human albumin; 1.0 to 5.0 parts by weight of ginseng stem and leaf extract; and 0.10 to 0.50 parts by weight of forest frog oil.

[0011] Furthermore, the composition further comprises an additive acceptable in the field of cosmetics. Preferably, the additive is selected from at least one of polydimethylsiloxane, hydrogenated lecithin, tragacanth gum, gum arabic, carboxymethyl cellulose, sodium alginate, polysorbate, carbomer, and phenoxyethanol.

[0012] Furthermore, the human albumin is selected from at least one of recombinant human albumin and blood-derived human albumin. Preferably, the human albumin is recombinant human albumin. Blood-derived human albumin refers to human albumin isolated and purified from healthy human blood, plasma, or specific immune plasma; and recombinant human albumin refers to human albumin prepared using recombinant DNA technology and used for the treatment and prevention of diseases.

[0013] A second aspect of the present invention provides use of the composition in preparing an anti-aging preparation. Preferably, the anti-aging preparation is a cosmetic. Preferably, the cosmetic comprises at least one of an emulsion, a cream, a toner, an essence, and a facial mask.

[0014] The third invention of the present invention provides a method for preparing the anti-aging composition, the method comprising the following steps:

[0015] (1) Ginseng stems and leaves were crushed and soaked in 75% to 80% (v / v) ethanol for 6 to 8 hours, the ethanol was removed by distillation, and then the mixture was suspended in deionized water and spray-dried to obtain ginseng stem and leaf extract spray-dried powder;

[0016] (2) adding the mass fraction of Rana sylvatica oil and the spray-dried powder of the ginseng stem and leaf extract obtained in step (1) to the mass fraction of human albumin aqueous solution or human albumin buffered saline solution, and homogenizing under high pressure at 500-1500N using a high-pressure homogenizer to obtain a mixed solution of the anti-aging composition, wherein the buffered saline solution includes at least one of phosphate buffer, citrate buffer, and acetate buffer, and preferably, the buffered saline solution is PBS buffer.

[0017] Furthermore, the inlet air temperature of the spray drying in step (1) is 120-160° C., the atomization pressure is 0.5-1.0 MPa, and the feed rate is 400-800 ml / h.

[0018] Furthermore, the method further comprises the step of adding additives acceptable in the field of cosmetics to the mixed solution obtained in step (2) and mixing the mixture.

[0019] In a specific embodiment of the present invention, the anti-aging preparation prepared from the anti-aging composition is an anti-aging facial cream, which comprises the following components: 5 to 20 parts by mass of human albumin; 1.0 to 5.0 parts by mass of ginseng stem and leaf extract; and 0.10 to 0.50 parts by mass of Rana sylvatica oil.

[0020] Furthermore, the anti-aging cream also includes additives acceptable in the cosmetics field. Preferably, the additives and their mass fractions include: 2.0-12.0 parts by mass of polydimethylsiloxane; 1.5-8.0 parts by mass of hydrogenated lecithin; 0.05-0.25 parts by mass of tragacanth; 0.05-0.30 parts by mass of gum arabic; 0.1-0.5 parts by mass of sodium carboxymethyl cellulose; 0.15-0.35 parts by mass of sodium alginate; 0.01-0.20 parts by mass of polysorbate; 0.05-0.40 parts by mass of carbomer; and 0.01-0.1 parts by mass of phenoxyethanol.

[0021] The present invention also provides a method for preparing the anti-aging cream, which comprises the following steps:

[0022] A. The ginseng stem and leaf extract is prepared by the following method: the ginseng stems and leaves used in the recipe are crushed and passed through a 10-mesh sieve to obtain a powder. The powder is then soaked in 75% ethanol at a solid-liquid ratio of 1:5 for 6 hours. The powder is then refluxed in a water bath for 15 minutes, repeated three times. The filtrates are combined and the ethanol is removed by distillation until no alcohol odor is present. The ginseng stem and leaf extract is then suspended in deionized water to a volume of 100 ml. The suspended solution is then spray-dried to obtain the ginseng stem and leaf extract.

[0023] B. Spray drying air inlet temperature: 120~160℃, atomizing pressure: 0.5~1.0MPa, feed rate: 400~800ml / h.

[0024] C. Stir hydrogenated lecithin, gum arabic, gum tragacanth, and polydimethylsiloxane in proportion by mass at 40-80 rpm, a temperature of 50° C.-90° C., and a time of 15-50 min to obtain phase A; stir sodium alginate, sodium carboxymethyl cellulose, frog oil, polysorbate, phenoxyethanol, and carbomer in proportion by mass at 35-80 rpm, a temperature of 55° C.-85° C., and a time of 15-45 min to obtain phase B; spray-dry phase A and phase B, as well as proportions of ginseng stem and leaf extract, to proportions of human albumin aqueous solution or human albumin PBS buffer, and homogenize at a high pressure of 500-1500 N for 30 min at a temperature of 25° C.-50° C. to obtain a mixed solution.

[0025] D. Place the mixed solution after high-pressure homogenization in a -4°C refrigerator for 24 hours to obtain the anti-aging cream containing human albumin.

[0026] The beneficial effects of the present invention include but are not limited to:

[0027] 1. The present invention provides an anti-aging composition containing human albumin, which uses human albumin as the main raw material. Through the synergistic enhancement of human albumin, ginseng stem and leaf extract, and forest frog oil, it can effectively promote the growth of fibroblasts, has a significant inhibitory effect on collagenase, and has a significant anti-wrinkle effect. It can effectively resist aging. Compared with existing cosmetics, it has simpler ingredients and better biocompatibility.

[0028] 2. The present invention also provides a method for preparing an anti-aging composition containing human albumin, which has a simple production process and great application prospects. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0030] Definition: Recombinant human albumin refers to human albumin prepared using recombinant DNA technology for treatment and prevention;

[0031] Hematogenous human albumin refers to human albumin separated and purified from healthy human blood, plasma or specific immune plasma.

[0032] Source of materials: Recombinant human albumin: Tonghua Anrui Biopharmaceutical Co., Ltd., batch number: ART101S;

[0033] Ginseng stems and leaves were purchased from Kangmei Xinkaihe (Jilin) ​​Pharmaceutical Co., Ltd., batch number: 20231120;

[0034] Rana sylvatica oil was provided by Fuzhentang (Changchun) Ginseng and Deer Antler Co., Ltd.

[0035] equipment:

[0036] Water bath (HH.S21-8, Shanghai Boxun Medical Biological Instrument Co., Ltd.);

[0037] Multifunctional fluorescence microplate reader (SPMax 3500FL, Shanghai Shanpu Biotechnology Co., Ltd.);

[0038] Analytical balance (BSA224S, Sartorius Scientific Instruments Beijing Co., Ltd.);

[0039] pH meter (PB-10, Sartorius Scientific Instruments Beijing Co., Ltd.);

[0040] CCK-8 kit (Lot No. 40203ES60, Yisheng Biotechnology (Shanghai) Co., Ltd.);

[0041] Human fibroblasts (Lot number: CBP61613, Nanjing Kebai Biotechnology Co., Ltd.);

[0042] Fibroblast medium-serum-free FM-sf (Cat. No. 2311, Beijing Yuhengfeng Technology Co., Ltd.);

[0043] Collagenase (lot number: C128695-2KU, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0044] Facial image analysis: VISIA image analysis software (Canfied, USA);

[0045] Cutometer (MPA580, CK, Germany) for skin elasticity testing;

[0046] Skin microscope and active skin surface analysis system VisioScan (VC20plus, CK Company, Germany);

[0047] Unless otherwise specified, other reagents and instruments used were commercially available.

[0048] Example 1

[0049] The preparation of the anti-aging composition containing human albumin comprises the following components in parts by weight: the effective ingredients are shown in Table 1,

[0050] Table 1 Components of various anti-aging compositions containing human albumin

[0051]

[0052] Table 2 Information of the control group

[0053]

[0054] The anti-aging composition was prepared according to the component information in Table 1. The specific method is as follows:

[0055] Step 1: Ginseng stems and leaves are crushed and soaked in 75% (v / v) ethanol for 6 hours, the ethanol is removed by distillation, and then the mixture is suspended in deionized water and spray-dried to obtain a ginseng stem and leaf extract spray-dried powder, wherein the spray drying has an inlet air temperature of 120-160°C, an atomization pressure of 0.5-1.0 MPa, and a feed rate of 400-800 ml / h;

[0056] Step 2: Add the mass fractions of Rana sylvatica oil and the spray-dried powder of the ginseng stem and leaf extract obtained in the above step 1 to the mass fractions of human albumin aqueous solution or human albumin PBS buffer, and use a high-pressure homogenizer at 500~1500N to obtain a mixed solution of the anti-aging composition.

[0057] Test example: Cell activity test

[0058] Based on the T / ZHCA021-2022 standard, this experiment used the CCK8 method for optimization. The effects of a recombinant human albumin anti-aging composition (groups 1-32), a commercial product (groups 33-34), and a blank control group (containing only cells, culture medium, and CCK-8 solution, group 35) on fibroblast viability were determined: cells in a good logarithmic growth phase were cultured at 5×10 3 Cells were seeded at a density of 100 μl / 100 μl in a 96-well plate and incubated overnight at 37°C in a 5% CO2 incubator. Groups (1-17) were added to the treated cells and cultured for 24 hours. 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for another 1 hour. OD values ​​were then measured. 450nm Calculate cell viability using the OD 450nm Value, recorded as As, control group and blank group OD 450nm The values ​​were recorded as Ac and Ab, and all samples were measured three times. Cell activity was calculated according to the following formula.

[0059] The experimental data are shown in Table 3.

[0060] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%

[0061] As: absorbance of the experimental group (containing cells, culture medium, CCK-8 solution and anti-aging composition);

[0062] Ac: absorbance of the control group (containing cells, culture medium, and CCK-8 solution, but not the anti-aging composition);

[0063] Ab: absorbance of the blank group (containing culture medium and CCK-8 solution, but not cells or anti-aging composition).

[0064] Table 3 Cell activity test results

[0065]

[0066] Table 3 shows the effect of a recombinant human albumin anti-aging composition on fibroblast viability. The effects of the prepared groups (1-10) on human fibroblast viability were investigated. The results showed that cell viability was above 110%, with the recombinant human albumin groups (1-5) exhibiting a superior effect compared to the hematopoietic human albumin groups (6-10), with cell viability exceeding 120%. This indicates that the prepared recombinant human albumin anti-aging composition is non-toxic to cells. In contrast, the blank control group (containing only cells, culture medium, and CCK-8 solution, group 18) had a cell viability of 100%, indicating that the anti-aging composition prepared by this method exhibited significant differences compared to the blank control, indicating that the recombinant human albumin anti-aging composition promotes fibroblast proliferation.

[0067] At the same time, the experimental data of the experimental groups (Groups 11-18) that only added recombinant human albumin, ginseng stem and leaf extract, and Rana sylvatica oil showed the lowest cell survival rate compared with the experimental groups (Groups 1-10, Groups 19-32) and the commercial group (Groups 33-34), indicating that the single component has the weakest cell proliferation activity. The experimental groups (24-32) that added only two of the three functional ingredients showed lower data than the commercial group (Groups 33-34), indicating that the anti-aging composition can maintain high cell activity under the premise of the coexistence of the three groups of active ingredients. The experimental group (Group 23) that added plant-derived protein at the optimal ratio showed lower data than the experimental groups (Groups 1-10), indicating that recombinant human albumin is superior to plant-derived albumin in promoting cell proliferation. At the optimal formula ratio, the experimental groups (Groups 1-10) were much higher than the other groups (11-34), with cell activity values ​​>110%, indicating that the heavy human albumin anti-aging composition prepared by this method has strong activity on human fibroblast growth.

[0068] Test Example Collagenase Inhibition Assay

[0069] The collagenase activity inhibition experiment was based on the experimental method established by Widowat et al. (Journal of Natural Remedies, 2016, 16 (3): 88-99), with some modifications. In a 96-well plate, 50 μl of the experimental group (Groups 1-32) and the commercial group (Groups 33-34) solution, 50 μl of collagenase solution (200 U / ml), and 50 μl of Tris-HCl buffer (0.05 mol / L, pH 7.5) were added sequentially. After vortexing and mixing, the mixture was incubated at 37°C for 15 minutes. Then, 50 μl of FALGPA (0.50 mg / ml) solution was added. The reaction was allowed to proceed at 37°C for 20 minutes, and the absorbance was measured at 350 nm. All samples were measured three times. The results are shown in Table 4.

[0070] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1-A BLANK / A TEST )×100% where:

[0071] A TEST A is the absorbance of the solution after all substances are mixed; BLANK It is the absorbance of the solution after mixing without adding the remaining sample substances.

[0072] Table 4 Collagenase inhibition test results

[0073]

[0074] As shown in Table 4, the experimental results of the human albumin anti-aging composition inhibiting collagenase show that the collagenase inhibition rate is above 66%, indicating that the prepared human albumin anti-aging composition has an inhibitory effect on collagenase.

[0075] The experimental data of the experimental groups (groups 11-18) that only added human albumin, ginseng stem and leaf extract, and frog oil showed the lowest collagenase inhibition rate compared with the experimental groups (groups 1-10, groups 19-32) and the commercial group (groups 33-34), indicating that the single component has the weakest effect on promoting collagen growth; the data results of the experimental groups (24-32) containing two functional ingredients at the same time were lower than those of the commercial group (groups 33-34), indicating that the anti-aging composition can only maintain a strong enzyme inhibition rate under the premise of the coexistence of three groups of active ingredients; the data results of the experimental group (group 23) with the optimal proportion of plant-derived protein added were lower than those of the experimental groups (groups 1-10), indicating that recombinant albumin is superior to plant-derived albumin in inhibiting collagenase. The optimal formula ratio of the experimental group (groups 1 to 10) was much higher than that of the other groups (11 to 34), and the collagenase inhibition rate was >80%, indicating that the human albumin anti-aging composition prepared by this method has significant inhibitory activity against collagenase and can play an anti-aging role.

[0076] Test examples: Hygiene index determination experiment and heavy metal determination

[0077] According to the microbiological and heavy metal testing methods in the "Safety Technical Specifications for Cosmetics" (2015 edition), the total bacterial count, thermotolerant Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, molds, yeasts, and heavy metals in the prepared recombinant human albumin anti-aging compositions (Groups 1-10) were determined. Microbiological index data are shown in Table 5, and heavy metal testing results are shown in Table 6.

[0078] Table 5 Results of microbiological index determination in each group

[0079]

[0080] As shown in Table 5, the test results of various hygienic indicators of a human albumin anti-aging composition (groups: 1 to 10) all meet the requirements.

[0081] Table 6 Determination results of heavy metal indicators in each group

[0082]

[0083] As shown in Table 6, the heavy metal detection index test results of a human albumin anti-aging composition (groups: 1 to 10) all meet the requirements.

[0084] Test case: Human skin irritation test

[0085] According to the Technical Specifications for Safety of Cosmetics (2015 edition), human patch tests were conducted. The composition prepared in the experimental group (Group 3) was selected as the experimental product. Fifteen males and 15 females aged 20 to 30 were selected as subjects (without skin diseases and iatrogenic skin sensitivity). The patch tests were performed on the inner forearm and behind the ear of the subjects. For the behind-the-ear patch test, the test patch device was removed 30 minutes after the test. For the inner forearm patch test, the test patch device was removed 0.5, 12, and 24 hours after the test. After the test, the skin reaction was observed and evaluated with reference to Table 7.

[0086] Table 7 Evaluation criteria for skin patch test

[0087]

[0088] The experimental results showed that the patch test results of 30 subjects were all negative, which shows that the composition prepared by the invention patent has no obvious irritation and sensitization to the human body and has good safety performance.

[0089] Test example: Anti-wrinkle efficacy test

[0090] According to the "Safety Technical Specifications for Cosmetics" (2015 edition), 30 Chinese women aged 35 to 65 years were selected and all received the composition prepared in the experimental group (Group 3). The subjects applied the composition once daily after cleansing their faces, applying it to the cheeks and gently massaging until absorbed. All other anti-wrinkle skincare products were discontinued during the experimental period. Skin tests were conducted at 0, 2, and 4 weeks. Before testing, the skin was cleansed with water and then placed in a constant temperature and humidity chamber for 30 minutes. After 30 minutes, the participants underwent facial anti-wrinkle efficacy testing. A control group was used as a control. The skin wrinkle scores, skin roughness, and skin elasticity of all participants before and after use served as indicators for the control group. The test subjects' indicators after 2 and 4 weeks of use served as indicators for the experimental group. The results are shown in Table 8.

[0091] Table 8 Comparison of anti-aging indicators of the face and cheek (X±s, n=30)

[0092]

[0093] Before using the composition, the average skin roughness of 30 subjects was 2.86±0.17, which decreased to 2.09±0.26 after two weeks of use, and the average skin roughness was significantly improved; the skin elasticity R2 value and skin elasticity Q1 value were 47.15±0.21 and 43.21±0.49, respectively, before use, and increased to 55.19±0.19 and 50.13±0.17, respectively, after two weeks of use. The skin elasticity values ​​showed an upward trend. The test data showed that the composition prepared by this method can effectively improve skin elasticity, make the skin firm, and thus have an anti-aging effect.

[0094] Example 2 Preparation of anti-aging facial cream containing recombinant human albumin comprising the following components in parts by mass: The active ingredients are shown in Table 9,

[0095] Table 9 Ingredients of various anti-aging creams containing recombinant human albumin

[0096]

[0097] The specific steps for preparing an anti-aging facial cream containing human albumin are as follows:

[0098] A second aspect of the present invention provides a method for preparing an anti-aging facial cream containing human albumin, comprising the following steps:

[0099] (1) The ginseng stem and leaf extract is prepared according to the following method: the ginseng stem and leaf used in the formula are crushed, passed through a 10-mesh sieve to obtain a powder, soaked in 75% ethanol at a solid-liquid ratio of 1:5 for 6 hours, reflux extracted at water bath temperature for 15 minutes, extracted three times, the filtrate is combined, and the ethanol is distilled off until there is no alcohol taste, the ginseng stem and leaf extract is suspended in purified water to 100 ml, the above solutions are mixed evenly, and the mixed solution is spray-dried to obtain the ginseng stem and leaf extract.

[0100] (2) Inlet air temperature for spray drying: 120-160°C, preferably 150°C; atomization pressure: 0.1-1.0 MPa, preferably 0.5 MPa; feed rate: 200-800 ml / h, preferably 450 ml / h.

[0101] (3) Stir hydrogenated lecithin, gum arabic, gum tragacanth and polydimethylsiloxane according to their mass fractions at 40-80 rpm, 50°C-90°C and 15-50 min to obtain phase A. Stir sodium alginate, sodium carboxymethyl cellulose, frog oil, polysorbate, phenoxyethanol and carbomer according to their mass fractions at 35-80 rpm, 55°C-85°C and 15-45 min to obtain phase B. Spray dry powder of phase A and phase B and ginseng stem and leaf extract by mass fractions are added to human albumin aqueous solution or human albumin PBS buffer by mass fractions, and homogenize with a high-pressure homogenizer at 500-1500N for 30 min at 25°C-50°C to obtain a mixed solution.

[0102] (4) The mixed solution after high-pressure homogenization is placed in a -4°C refrigerator for 24 hours to obtain the recombinant human albumin anti-aging cream.

[0103] Test example: Cell activity test

[0104] Based on the T / ZHCA021-2022 standard, this experiment used the CCK8 method for optimization. The effects of a recombinant human albumin anti-aging cream (groups 1 to 10) and a blank control group (containing only cells, culture medium, and CCK-8 solution, group 11) on fibroblast viability were determined: cells in good logarithmic growth phase were cultured at 5×10 3 Cells were seeded at a density of 100 μl / 100 μl in a 96-well plate and incubated overnight at 37°C in a 5% CO2 incubator. Groups (1-10) of cells were added and treated, respectively, and cultured for 24 hours. 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for another hour. OD values ​​were then measured. 450nm Calculate cell viability using the OD 450nm Value, recorded as As, OD of control group and blank group 450nm The values ​​were recorded as Ac and Ab, and all samples were measured three times. Cell activity was calculated according to the following formula.

[0105] The experimental data are shown in Table 10.

[0106] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%

[0107] As: absorbance of the experimental group (containing cells, culture medium, CCK-8 solution, and anti-aging cream);

[0108] Ac: absorbance of the control group (containing cells, culture medium, and CCK-8 solution, but not anti-aging cream);

[0109] Ab: absorbance of the blank group (containing culture medium and CCK-8 solution, but not cells or anti-aging cream).

[0110] Table 10 Cell activity test results

[0111]

[0112] As shown in Table 10, the effect of a human albumin anti-aging cream on fibroblast viability is similar to the results of groups 1-10 in Example 1. This demonstrates that the addition of other additives acceptable in the cosmetics field does not affect the effect of the anti-aging composition of the present invention, and that the anti-aging cream of the present invention has a good effect in promoting fibroblast survival.

[0113] Test Example Collagenase Inhibition Assay

[0114] The collagenase activity inhibition assay was based on the experimental method established by Widowati et al. (Journal of Natural Remedies, 2016, 16(3):88-99), with some modifications. In a 96-well plate, 50 μl of the experimental group (groups 1-10) solution, 50 μl of the collagenase solution (200 U / ml), and 50 μl of Tris-HCl buffer (0.05 mol / L, pH 7.5) were added sequentially. After vortexing and mixing, the mixture was incubated at 37°C for 15 minutes. Then, 50 μl of FALGPA (0.50 mg / ml) solution was added. The reaction was incubated at 37°C for 20 minutes, and the absorbance was measured at 350 nm. All samples were measured three times. The results are shown in Table 11.

[0115] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1-A BLANK / A TEST )×100% where:

[0116] A TEST A is the absorbance of the solution after all substances are mixed; BLANK It is the absorbance of the solution after mixing without adding the remaining sample substances.

[0117] Table 11 Collagenase inhibition test results

[0118]

[0119] As shown in Table 11, the composition of the present invention has no significant change in its inhibitory effect on collagenase after adding additives in the cosmetic field, and has a good collagenase inhibitory effect.

[0120] Test examples: Hygiene index determination experiment and heavy metal determination

[0121] According to the microbiological and heavy metal testing methods in the "Safety Technical Specifications for Cosmetics" (2015 edition), total colony count, thermotolerant Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, molds, yeasts, and heavy metals in a recombinant human albumin anti-aging cream (Groups 1-10) were determined. Microbiological index data are shown in Table 12, and heavy metal testing results are shown in Table 13.

[0122] Table 12 Results of microbiological index determination in each group

[0123]

[0124] As shown in Table 12, the test results of various hygienic indicators of a recombinant human albumin anti-aging cream (groups: 1-10) all meet the requirements.

[0125] Table 13 Determination results of heavy metal indicators in each group

[0126]

[0127] As shown in Table 13, the heavy metal detection index test results of a recombinant human albumin anti-aging cream (groups: 1-10) all meet the requirements.

[0128] Test case: Human skin irritation test

[0129] According to the Technical Specifications for Safety of Cosmetics (2015 edition), human patch tests were conducted. The cream prepared in the experimental group (Group 3) was selected as the experimental product. Fifteen males and 15 females aged 20 to 30 were selected as subjects (without skin diseases and iatrogenic skin sensitivity). The patch tests were performed on the inner forearm and behind the ear of the subjects. For the patch test behind the ear, the test patch device was removed 30 minutes after the test. For the patch test on the inner forearm, the test patch device was removed 0.5, 12, and 24 hours after the test. After the test, the skin reaction was observed and evaluated with reference to Table 14.

[0130] Table 14 Evaluation criteria for skin patch test

[0131]

[0132] The experimental results showed that the patch test results of 30 subjects were all negative, which shows that the composition prepared by the invention patent has no obvious irritation and sensitization to the human body and has good safety performance.

[0133] Test example: Anti-wrinkle efficacy test

[0134] According to the "Safety Technical Specifications for Cosmetics" (2015 edition), 30 Chinese women aged 35 to 65 were selected and all used the face cream prepared in the experimental group (Group 3). The subjects applied the cream once daily after cleansing their faces, applying it to the cheeks and gently massaging until absorbed. All other anti-wrinkle skincare products were discontinued during the experimental period. Skin tests were conducted at 0, 2, and 4 weeks. Before the tests, the skin was cleansed with water and then placed in a constant temperature and humidity chamber for 30 minutes. After 30 minutes, the participants underwent facial anti-wrinkle efficacy testing. A control group was used as a control. The skin wrinkle scores, skin roughness, and skin elasticity of all participants before and after use served as indicators for the control group. The indicators of the test subjects after 2 and 4 weeks of use served as indicators for the experimental group. The results are shown in Table 15.

[0135] Table 15 Comparison of anti-aging indicators of face and cheek (X±s, n=30)

[0136]

[0137] Table 15 shows that the facial cream prepared by this method can effectively improve skin elasticity and make the skin firm, and the effect is not much different from that of the composition without adding additives, and has a good anti-wrinkle effect.

[0138] The foregoing is merely an embodiment of the present invention. The scope of protection of the present invention is not limited by these specific embodiments but is determined by the claims of the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the technical concepts and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. An anti-aging composition containing recombinant human albumin, characterized in that: The composition comprises the following components in parts by weight: 5 to 20 parts by weight of recombinant human albumin; 1.0 to 5.0 parts by weight of ginseng stem and leaf extract; and 0.10 to 0.50 parts by weight of forest frog oil.

2. The composition according to claim 1, characterized in that The composition may further include additives acceptable in the cosmetic field.

3. The composition according to claim 2, characterized in that The additive is selected from at least one of polydimethylsiloxane, hydrogenated lecithin, tragacanth gum, gum arabic, carboxymethyl cellulose, sodium alginate, polysorbate, carbomer and phenoxyethanol.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of anti-aging preparations.

5. The use according to claim 4, characterized in that: The anti-aging preparation is a cosmetic.

6. The use according to claim 4, characterized in that: The anti-aging preparation comprises at least one of lotion, cream, toner, essence and facial mask.

7. A method for preparing the composition according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) After crushing the ginseng stems and leaves, soak them in 75% to 80% (v / v) ethanol for 6 to 8 hours, distill the ethanol off, and then suspend them in deionized water and spray dry them to obtain ginseng stem and leaf extract spray-dried powder; (2) adding 100 mass parts of Rana sylvatica oil and the spray-dried powder of ginseng stem and leaf extract obtained in step (1) to 100 mass parts of recombinant human albumin aqueous solution or recombinant human albumin buffered saline solution, and homogenizing them under high pressure at 500-1500 N using a high-pressure homogenizer to obtain a mixed solution of the anti-aging composition.

8. The method according to claim 7, characterized in that The inlet air temperature of the spray drying in step (1) is 120-160°C, the atomization pressure is 0.5-1.0 MPa, and the feed rate is 400-800 ml / h.

9. The method according to claim 7, characterized in that: The method further comprises the step of adding additives acceptable in the field of cosmetics to the mixed solution obtained in step (2) and mixing the mixture.

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