Anti-aging composition containing human albumin as well as preparation method and application of anti-aging composition

Abstract

The invention discloses an anti-aging composition containing human albumin as well as a preparation method and application of the anti-aging composition, and relates to the field of cosmetics. The mass part of the ginseng stem and leaf extract is 1.0-5.0 parts; and the mass part of the rana japonica oil is 0.10-0.50. The human albumin, the ginseng stem and leaf extract and the rana japonica oil have a synergistic effect, can effectively promote the growth of fibroblasts, have an obvious inhibition effect on collagenase, and have an obvious anti-wrinkle effect and a good anti-aging effect. The anti-aging composition disclosed by the invention is simple in component and simple in preparation method, and has a wide application prospect in the field of cosmetics.

Description

Technical Field

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

[0002] Human skin is an organ that easily ages. As we age, the aging characteristics of the skin also appear: more wrinkles, sagging and yellowing of the skin, reduced luster, luster and smoothness, coarsening of the texture, increased pigmentation or the appearance of depigmentation, etc. It is generally believed that the causes of skin aging are mainly endogenous factors and exogenous factors. Endogenous factors include: (1) genetic factors, because the order of chromosome polymerization produced by genetic inheritance will change with age; (2) hormone shortage, due to hormone deficiency, hormone aging occurs. For example, estrogen deficiency will reduce the function of the skin's connective tissue and cause various skin changes. Exogenous factors include: (1) pollution, namely, the pollution caused by the growing 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, pore blockage, 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, thinning of the epidermal dermal connective tissue that controls skin fine-tuning, etc. In addition, excessive ultraviolet exposure is also the main 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. Consumers' demand for safe and non-irritating cosmetics has promoted the application of natural active ingredients in cosmetics. Natural ingredients with physiological activities such as anti-free radical, anti-oxidation, anti-radiation, anti-bacterial, and anti-inflammatory are increasingly valued by people. At present, the development of natural active ingredients that can be used in anti-aging cosmetics has become a trend.

[0005] Among the existing anti-aging products, there are products that add albumin, but albumin appears more as an auxiliary functional component or acts together with other active polypeptides. For example, in a stem cell skin care composition disclosed in CN106983713A, and a preparation method and a use method thereof, the content of serum albumin is only 1% of the main effective ingredient, the stem cell extract; in an anti-aging composition disclosed in CN110613628A, a lyophilized powder containing the anti-aging composition, and a preparation method thereof, serum albumin is compounded with specific oligopeptides to produce an anti-aging effect, but serum albumin is more used as a protein protective agent. At present, there are relatively few reports on anti-aging products using serum albumin as the main effective ingredient for antioxidants. 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 forest frog oil.

[0007] Human serum albumin (HSA) is a monomeric protein produced by the liver. Mature human serum albumin contains 585 amino acid residues and has a molecular weight of 66KD. Albumin has abundant free sulfhydryl groups and antioxidant properties. It can scavenge free radicals (such as hypochlorous acid (HOCl) and peroxynitrite (ONOOH)) and provide sulfhydryl groups (-SH) to produce anti-aging effects.

[0008] Ginseng stems and leaves: This product is the dried stems and leaves of Panax ginseng CA Mey., a plant of the Araliaceae family. Ginseng stems and leaves are cold in nature, bitter and sweet in taste. They belong to the lung meridian and stomach meridian. They have the effects of replenishing qi, benefiting the lungs, removing heat, and promoting body fluid. Current pharmacological studies have shown that ginsenosides, which are rich in ginseng stems and leaves, have certain antioxidant and anti-inflammatory effects and can delay cell aging. The ginseng polysaccharides contained in them have a positive effect on enhancing immune function and promoting hematopoiesis.

[0009] Rana oil is obtained by drying the oviduct of the female Chinese frog (Rana temporariachensinensis David), a species of the Ranidae family. It tastes sweet and salty, is neutral in nature, and enters the lung and kidney meridians. It has the effects of nourishing the kidney and essence, nourishing yin and moistening the lungs. Scientific research shows that Rana oil is rich in bioactive peptides, which are effective in regulating the synthesis of collagen, elastin and melanin in dermatology, and have 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 also includes 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 hematogenous human albumin. Preferably, the human albumin is recombinant human albumin. Hematogenous human albumin refers to human albumin separated and purified from healthy human blood, plasma or specific immune plasma; recombinant human albumin refers to human albumin prepared by recombinant DNA technology for the treatment and prevention of diseases.

[0013] In a second aspect of the present invention, the use of the composition in preparing an anti-aging preparation is provided. Preferably, the anti-aging preparation is a cosmetic. Preferably, the cosmetic comprises at least one of an emulsion, a cream, a lotion, 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: (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 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 comprises at least one of a phosphate buffer, a citrate buffer, and an acetate buffer, and preferably, the buffered saline solution is a PBS buffer.

[0015] 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.

[0016] Furthermore, the method further comprises the step of adding an additive acceptable in the field of cosmetics to the mixed solution obtained in step (2) and mixing the mixture.

[0017] In a specific embodiment of the present invention, the anti-aging preparation prepared by the anti-aging composition is an anti-aging 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 forest frog oil.

[0018] Furthermore, the anti-aging cream also includes additives acceptable in the field of cosmetics. 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.

[0019] The present invention also provides a method for preparing the anti-aging cream, which comprises the following specific steps: A. The ginseng stem and leaf extract is prepared according to the following method: crush the ginseng stem and leaf used in the formula, pass through a 10-mesh sieve to obtain powder, soak it in 75% ethanol at a solid-liquid ratio of 1:5 for 6 hours, reflux extract for 15 minutes at a water bath temperature, extract 3 times, combine the filtrate, distill to remove the ethanol until there is no alcohol taste, add deionized water to suspend the ginseng stem and leaf extract to 100 ml, spray dry the suspended solution, and obtain the ginseng stem and leaf extract.

[0020] B. Inlet air temperature for spray drying: 120~160℃, atomization pressure: 0.5~1.0Mpa, feed rate: 400~800ml / h.

[0021] C. Stir hydrogenated lecithin, gum arabic, gum tragacanth and polydimethylsiloxane according to their mass fractions at 40-80 rpm, at a temperature of 50°C-90°C, for 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, at a temperature of 55°C-85°C, for 15-45 min to obtain phase B; spray-dry phase A and phase B and a mass fraction of ginseng stem and leaf extract, add a mass fraction of human albumin aqueous solution or human albumin PBS buffer, and homogenize with a high-pressure homogenizer at 500-1500N for 30 min at a temperature of 25°C-50°C to obtain a mixed solution.

[0022] 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.

[0023] The beneficial effects of the present invention include but are not limited to: 1. The present invention provides an anti-aging composition containing human albumin, which takes human albumin as the main raw material. Through the synergistic enhancement of human albumin, ginseng stem and leaf extract, and forest frog oil, the composition can effectively promote the growth of fibroblasts, has a significant inhibitory effect on collagenase, and has a significant anti-wrinkle effect, and can effectively resist aging. Compared with existing cosmetics, the composition is simple and the biocompatibility is better.

[0024] 2. The present invention also provides a method for preparing an anti-aging composition containing human albumin, which has a simple production process and has high application prospects. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to 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.

[0026] Definition: Recombinant human albumin refers to human albumin prepared using recombinant DNA technology for treatment and prevention; Hematogenous human albumin refers to human albumin separated and purified from healthy human blood, plasma or specific immune plasma.

[0027] Source of materials: Recombinant human albumin: Tonghua Anrui Special Biopharmaceutical Co., Ltd., batch number: ART101S; Ginseng stems and leaves were purchased from Kangmei Xinkaihe (Jilin) ​​Pharmaceutical Co., Ltd., batch number: 20231120; Rana affine oil was provided by Fuzhentang (Changchun) Ginseng and Deer Antler Co., Ltd.

[0028] equipment: Water bath (HH.S21-8, Shanghai Boxun Medical Biological Instrument Co., Ltd.); Multifunctional fluorescence microplate reader (SPMax 3500FL, Shanghai Shanpu Biotechnology Co., Ltd.); Analytical balance (BSA224S, Sartorius Scientific Instruments Beijing Co., Ltd.); pH meter (PB-10, Sartorius Scientific Instruments Beijing Co., Ltd.); CCK-8 kit (Batch No.: 40203ES60, Yisheng Biotechnology (Shanghai) Co., Ltd.); Human fibroblasts (Lot No.: CBP61613, Nanjing Kebai Biotechnology Co., Ltd.); Fibroblast medium-serum-free FM-sf (Cat. No. 2311, Beijing Yuhengfeng Technology Co., Ltd.); Collagenase (lot number: C128695-2KU, Shanghai Aladdin Biochemical Technology Co., Ltd.); Facial image analysis: VISIA image analysis software (Canfied, USA); Skin elasticity test probe Cutometer (MPA580, CK Company, Germany); Skin microscope and active skin surface analysis system VisioScan (VC20plus, CK Company, Germany); Unless otherwise specified, other reagents and instruments used were commercially available.

[0029] Example 1 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, Table 1 Components of various anti-aging compositions containing human albumin

[0030] Table 2 Information of control group

[0031] The anti-aging composition was prepared according to the component information in Table 1, and the specific method was as follows: Step 1: After crushing the ginseng stems and leaves, soak them in 75% (v / v) ethanol for 6 hours, distill to remove the ethanol, and then suspend them in deionized water and spray dry them to obtain ginseng stem and leaf extract spray-dried powder, wherein the spray drying air inlet temperature is 120-160°C, the atomization pressure is 0.5-1.0Mpa, and the feed rate is 400-800ml / h; Step 2: Add the mass fraction 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 fraction 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.

[0032] Test case Cell activity test Based on the T / ZHCA021-2022 standard, this experiment used the CCK8 method for corresponding optimization. The effects of a recombinant human albumin anti-aging composition (groups 1-32), commercial products (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 good logarithmic growth phase were cultured at 5×10 3The cells were inoculated at a density of 100 μl / 100 μl in a 96-well plate and incubated overnight at 37°C in a 5% CO2 constant temperature incubator. The cells were treated with groups (1-17) and cultured for 24 hours. 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for 1 hour. The OD was measured. 450nm The cell viability was calculated 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.

[0033] The experimental data are shown in Table 3.

[0034] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100% As: absorbance of the experimental group (containing cells, culture medium, CCK-8 solution and anti-aging composition); Ac: absorbance of the control group (containing cells, culture medium, CCK-8 solution, but not the anti-aging composition); Ab: absorbance of blank group (containing culture medium and CCK-8 solution, but not containing cells or anti-aging composition).

[0035] Table 3 Cell activity test results

[0036] As shown in Table 3, the effect of a recombinant human albumin anti-aging composition on the viability of fibroblasts: the effect on the viability of human fibroblasts was explored through the prepared groups (1-10), and the results showed that the cell survival rate was above 110%, and the recombinant human albumin group (1-5) had a better effect than the hematogenous human albumin group (6-10), with a cell survival rate of above 120%. This shows that the prepared recombinant human albumin anti-aging composition is non-toxic to cells, while the cell survival rate of the blank control group (containing only cells, culture medium, CCK-8 solution, group 18) is 100%, indicating that the anti-aging composition prepared by this method has a significant difference compared with the blank control, indicating that the recombinant human albumin anti-aging composition has the effect of promoting cell proliferation on fibroblasts.

[0037] 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 forest frog oil showed the lowest cell survival rate compared with the experimental groups (groups 1-10, 19-32) and the commercial group (groups 33-34), indicating that the single component had the weakest cell proliferation activity; at the same time, the experimental groups (24-32) that only added two of the three functional ingredients had data results lower 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 according to the optimal ratio had data results lower than the experimental group (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 other groups (11-34), and the cell activity value was >110%, indicating that the heavy human albumin anti-aging composition prepared by this method has strong activity on the growth of human fibroblasts.

[0038] Test Example Collagenase Inhibition Assay The collagenase activity inhibition experiment was modified based on the experimental method established by Widowat.W (Journal of Natual Remedies, 2016, 16 (3): 88-99). In a 96-well plate, 50 μl of the experimental group (groups 1-32) and the commercial group (groups 33-34), 50 μl of collagenase solution (200 U / ml) and 50 μl of Tris-HCl buffer (0.05 mol / L, pH 7.5) were added in turn. After shaking and mixing, the mixture was incubated at 37°C for 15 minutes, and 50 μl of FALGPA (0.50 mg / ml) solution was added. The mixture was reacted at 37°C for 20 minutes, and the absorbance was measured at 350 nm. All samples were measured three times. The data results are shown in Table 4.

[0039] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1-A BLANK / A TEST )×100%Where: 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.

[0040] Table 4 Collagenase inhibition test results

[0041] 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.

[0042] 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, 19-32) and the commercially available groups (groups 33-34), indicating that a 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 commercially available groups (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) that added plant-based protein in the optimal ratio were lower than those of the experimental groups (groups 1-10), indicating that recombinant albumin is superior to plant-based albumin in its ability to inhibit collagenase. The optimal formula ratio of the experimental group (groups 1-10) was much higher than that of other groups (11-34), and the collagenase inhibition rate was >80%, indicating that the human albumin anti-aging composition prepared by this method has significant inhibitory effect on collagenase and can play an anti-aging role.

[0043] Test examples: Hygiene index determination experiment and heavy metal determination According to the microbial and heavy metal testing methods of the "Safety Technical Specifications for Cosmetics" (2015 edition), the total colony count, heat-resistant Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, molds, yeasts and heavy metals in the prepared recombinant human albumin anti-aging composition (groups 1 to 10) were determined. The microbiological index data are shown in Table 5, and the heavy metal detection test results are shown in Table 6.

[0044] Table 5 Results of microbiological index determination in each group

[0045] As can be seen from Table 5, the test results of various hygienic indicators of a human albumin anti-aging composition (groups: 1-10) all meet the requirements.

[0046] Table 6 Determination results of heavy metal indicators in each group

[0047] As can be seen from Table 6, the heavy metal detection index test results of a human albumin anti-aging composition (groups: 1-10) all meet the requirements.

[0048] Test Case Human Skin Irritation Test According to the Technical Specification 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. 15 males and 15 females aged 20-30 were selected as subjects (without skin diseases and iatrogenic skin sensitivity). The patch tests were conducted 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 7.

[0049] Table 7 Evaluation criteria for skin patch test

[0050] The experimental results showed that the patch test results of the 30 subjects were all negative, which indicated that the composition prepared by the invention patent had no obvious irritation and sensitization to the human body and had good safety performance.

[0051] Test case: Anti-wrinkle efficacy test According to the "Safety Technical Specifications for Cosmetics" (2015 edition), the subjects were screened and 30 Chinese women aged 35 to 65 years old were selected. All of them used the composition prepared by the experimental group (Group 3). The subjects used it once a day after cleansing their faces, applied it on the cheeks of the face, and gently massaged until absorbed. During the experiment, other anti-wrinkle skin care products were stopped. Skin tests were conducted at week 0, 2 weeks, and 4 weeks. Before the test, the facial skin was cleaned with clean water. After cleaning, the subjects sat in a constant temperature and humidity room for 30 minutes. After 30 minutes, the facial skin anti-wrinkle efficacy test was conducted on the subjects participating in the experiment, and the control was selected as the control before and after. The skin wrinkle scores, skin roughness, and skin elasticity test data of all subjects before using the product were used as the control group indicators, and the indicators of the test subjects after using the product for 2 weeks and 4 weeks were used as the experimental group indicators. The experimental results are shown in Table 8.

[0052] Table 8 Comparison of anti-aging indicators of face and cheek (X±s, n=30)

[0053] 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 the skin elasticity Q1 value were 47.15±0.21 and 43.21±0.49 before use, respectively, and increased to 55.19±0.19 and 50.13±0.17 after two weeks of use, respectively, and the skin elasticity values ​​showed an upward trend. The test data show that the composition prepared by this method can effectively improve skin elasticity and make the skin firm, thereby playing an anti-aging effect.

[0054] Example 2 Preparation of anti-aging facial cream containing recombinant human albumin comprising the following components in parts by weight: The effective ingredients are shown in Table 9, Table 9 Ingredients of various anti-aging creams containing recombinant human albumin

[0055] The specific steps of preparing an anti-aging facial cream containing human albumin are as follows: The second aspect of the present invention provides a method for preparing an anti-aging facial cream containing human albumin, comprising the following steps: (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, refluxed for 15 minutes at a water bath temperature, extracted three times, the filtrates are 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.

[0056] (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.

[0057] (3) Stir hydrogenated lecithin, gum arabic, gum tragacanth and polydimethylsiloxane in proportion by mass at 40-80 rpm, at a temperature of 50°C-90°C, for 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, at a temperature of 55°C-85°C, for 15-45 min to obtain phase B; spray-dry phase A and phase B and a proportion by mass of ginseng stem and leaf extract into a proportion by mass of human albumin aqueous solution or human albumin PBS buffer, and homogenize with a high-pressure homogenizer at 500-1500 N for 30 min at a temperature of 25°C-50°C to obtain a mixed solution.

[0058] (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.

[0059] Test case Cell activity test Based on the T / ZHCA021-2022 standard, this experiment used the CCK8 method for corresponding optimization. The effect 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 the viability of fibroblasts was determined: cells in good logarithmic growth phase were cultured at 5×10 3 The cells were inoculated at a density of 100 μl / 100 μl in a 96-well plate and incubated overnight at 37°C in a 5% CO2 constant temperature incubator. The cells were treated with groups (1-10) and cultured for 24 h. 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for 1 h. The OD was measured. 450nm The cell viability was calculated 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.

[0060] The experimental data are shown in Table 10.

[0061] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100% As: absorbance of the experimental group (containing cells, culture medium, CCK-8 solution, and anti-aging cream); Ac: absorbance of the control group (containing cells, culture medium, CCK-8 solution, but not anti-aging cream); Ab: absorbance of blank group (containing culture medium and CCK-8 solution, but not containing cells or anti-aging cream).

[0062] Table 10 Cell activity test results

[0063] As can be seen from Table 10, the effect of a human albumin anti-aging cream on the viability of fibroblasts: compared with the results of groups 1-10 in Example 1, its effect on the viability of fibroblasts is not much different, which can prove that after adding other additives acceptable in the field of cosmetics, it does not affect the effect of the anti-aging composition of the present invention, and the anti-aging cream of the present invention has a good effect of promoting the survival of fibroblasts. Test Example Collagenase Inhibition Assay The collagenase activity inhibition experiment was modified based on the experimental method established by Widowati.W (Journal of NatualRemedies, 2016, 16 (3): 88-99). In a 96-well plate, 50 μl of the experimental group (groups 1-10), 50 μl of collagenase solution (200 U / ml) and 50 μl of Tris-HCl buffer (0.05 mol / L, pH 7.5) were added in sequence, and after oscillation and mixing, the mixture was incubated at 37°C for 15 min, and 50 μl of FALGPA (0.50 mg / ml) solution was added. The mixture was reacted at 37°C for 20 min, and the absorbance was measured at 350 nm. All samples were measured three times. The data results are shown in Table 11.

[0064] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1-A BLANK / A TEST )×100%Where: 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.

[0065] Table 11 Collagenase inhibition test results

[0066] It can be seen from Table 11 that after the composition of the present invention is added with additives in the field of cosmetics, its inhibitory effect on collagenase does not change significantly, and it has a good collagenase inhibitory effect. Test examples: Hygiene index determination experiment and heavy metal determination According to the microbiological and heavy metal test methods of the "Safety Technical Specifications for Cosmetics" (2015 edition), the total colony count, heat-resistant Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, molds, yeasts and heavy metals in a recombinant human albumin anti-aging cream (groups 1-10) were determined. The microbiological index data are shown in Table 12, and the heavy metal test results are shown in Table 13.

[0067] Table 12 Results of microbiological index determination in each group

[0068] It can be seen from Table 12 that the test results of various indicators in hygiene of a recombinant human albumin anti-aging cream (groups: 1-10) are in compliance with the regulations.

[0069] Table 13 Determination results of heavy metal indicators in each group

[0070] It can be seen from Table 13 that the heavy metal detection index test results of a recombinant human albumin anti-aging cream (groups: 1-10) all meet the requirements.

[0071] Test Case Human Skin Irritation Test According to the Technical Specification 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. 15 males and 15 females aged 20-30 were selected as subjects (without skin diseases and iatrogenic skin sensitivity). The patch tests were conducted 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.

[0072] Table 14 Evaluation criteria for skin patch test

[0073] The experimental results showed that the patch test results of the 30 subjects were all negative, which indicated that the composition prepared by the invention patent had no obvious irritation and sensitization to the human body and had good safety performance.

[0074] Test Case: Anti-wrinkle efficacy test According to the "Safety Technical Specifications for Cosmetics" (2015 edition), the subjects were screened and 30 Chinese women aged 35 to 65 years old were selected. All of them used the cream prepared by the experimental group (Group 3). The subjects used it once a day after cleansing their faces, applied it on the cheeks of the face, and gently massaged until absorbed. During the experiment, other anti-wrinkle skin care products were stopped. Skin tests were conducted at week 0, 2 weeks, and 4 weeks. Before the test, the facial skin was cleaned with clean water. After cleaning, the subjects sat in a constant temperature and humidity room for 30 minutes. After 30 minutes, the facial skin anti-wrinkle efficacy test was conducted on the subjects participating in the experiment, and the control was selected as the control before and after. The skin wrinkle score, skin roughness, and skin elasticity test data of all subjects before using the product were used as the control group indicators, and the indicators of the test subjects after using the product for 2 weeks and 4 weeks were used as the experimental group indicators. The experimental results are shown in Table 15.

[0075] Table 15 Comparison of anti-aging indicators of face and cheek (X±s, n=30)

[0076] 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.

[0077] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-aging composition containing human albumin, characterized in that: 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.

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. The composition according to claim 1, characterized in that The human albumin is selected from at least one of recombinant human albumin and hematogenous human albumin.

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

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

7. The use according to claim 5, characterized in that: The anti-aging preparation comprises at least one of lotion, cream, toner, essence and facial mask.

8. A method for preparing the composition according to any one of claims 1 to 4, 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 10 parts by weight of Rana sylvatica oil and the spray-dried powder of ginseng stem and leaf extract obtained in step (1) to 10 parts by weight of human albumin aqueous solution or human albumin buffered saline solution, and homogenizing them under high pressure at 500-1500N using a high-pressure homogenizer to obtain a mixed solution of the anti-aging composition.

9. The method according to claim 8, 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.

10. The method according to claim 8, 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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