Dendrobium officinale small-molecule active peptide with anti-oxidation and anti-aging effects

Abstract

The invention provides a dendrobium officinale micromolecular active peptide with antioxidant and anti-aging effects, and belongs to the technical field of cosmetic raw material development. The invention proves that the dendrobium officinale polypeptide has an excellent application prospect in serving as a cosmetic raw material. According to the embodiment of the invention, natural wild dendrobium officinale with high quality and rich active ingredients is selected as an extraction raw material of the plant polypeptide, and the molecular weight of the dendrobium officinale polypeptide is measured to determine that the dendrobium officinale polypeptide belongs to the micromolecular polypeptide. The application effect of the dendrobium officinale polypeptide in cosmetics is evaluated through in-vitro cell experiments, and it is proved that the dendrobium officinale polypeptide can promote expression of collagen related genes, inhibit expression of collagen degrading enzyme related genes and increase the collagen content and has the anti-aging and tightening effects; the expression of inflammatory factor related genes is inhibited, the occurrence of immune inflammation reaction is reduced, and an anti-inflammatory effect is achieved; the ROS content is inhibited, the oxidative stress is reduced, and the antioxidant effect is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic raw material development, and in particular to a Dendrobium officinale small molecule active peptide with anti-oxidation and anti-aging effects. Background Art

[0002] Peptides are compounds formed by α-amino acids linked together by peptide bonds and are intermediate products of protein hydrolysis. According to the differences in the relative molecular weight of polypeptides, polypeptides are roughly divided into: ① Oligopeptides: also known as small molecule polypeptides, the relative molecular weight is usually distributed between a few hundred and a few thousand Daltons (Da), usually composed of 2 to 20 amino acids; ② Polypeptides: The relative molecular weight is distributed between a few thousand and 20,000 Daltons, usually composed of 20 to 100 amino acids; ③ Proteins: The molecular weight is usually higher than 20,000 Daltons, with a complex tertiary or quaternary higher-level structure. Due to their small molecular weight, oligopeptides are better in water solubility and easier to absorb, so many current active pharmaceutical ingredients are small molecule polypeptides.

[0003] At present, there are also a variety of polypeptide products in cosmetic raw materials. For example, Chinese patent CN112842927A discloses a plant-derived polypeptide and cosmetics containing the plant-derived polypeptide, which include ACEI peptides, antimicrobial peptides and morphine-like antagonist peptides. The extraction of the ACEI peptide includes the following important ingredients: 1-3 parts of soybeans and 2-5 parts of corn; the extraction of the antimicrobial peptide includes the following important ingredients: 2-6 parts of hibiscus flowers, 1-4 parts of chrysanthemums, 1-6 parts of white poria cocos and 1-3 parts of angelica; the extraction of the morphine-like antagonist peptide includes the following important ingredients: 1-3 parts of olives, 1-4 parts of red pomegranates and 1-3 parts of blueberries.

[0004] However, this solution has complicated raw materials and questionable safety performance, is not suitable for industrial production, and its efficacy needs to be improved. Summary of the invention

[0005] The purpose of the present invention is to provide a small molecule active peptide of Dendrobium officinale with anti-oxidation and anti-aging effects, which has excellent performance as a cosmetic raw material and has broad application prospects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an application of a Dendrobium officinale polypeptide in the preparation of cosmetics.

[0007] Preferably, the cosmetic is an anti-aging skin product.

[0008] Preferably, the cosmetic is an anti-inflammatory product.

[0009] Preferably, the cosmetic is an antioxidant product.

[0010] Preferably, the Dendrobium officinale polypeptide is obtained by a preparation method comprising the following steps: freeze-drying and pulverizing the Dendrobium officinale in sequence to obtain Dendrobium officinale powder; The powder of Dendrobium officinale is mixed with a buffer solution containing NaCl, and subjected to freeze-thaw treatment to obtain an intermediate; The intermediate is placed at -5 to 5°C for 5 to 20 minutes, and then subjected to heating treatment and centrifugation treatment in sequence to obtain a supernatant; The supernatant is freeze-dried to obtain the Dendrobium officinale polypeptide.

[0011] Preferably, the Dendrobium officinale is pre-frozen before freeze-drying.

[0012] Preferably, the pre-freezing temperature is -40 to -20°C; The pre-freezing time is 12 to 36 hours.

[0013] Preferably, the cold trap temperature of the freeze-drying is -100 to -60°C; The freezer temperature of the freeze-drying chamber is -45°C to 35°C; The vacuum degree of the freeze drying is 0-10 Pa; The freeze-drying time is 36 to 72 hours.

[0014] Preferably, the solid-liquid ratio of the Dendrobium officinale powder and the NaCl-containing buffer solution is 1:5-10; The buffer is PBS; The final concentration of NaCl in the buffer is 100-200 mM; The freezing stage in the freeze-thaw treatment adopts liquid nitrogen freezing; The temperature used in the thawing stage of the freeze-thaw treatment is 35-40°C; The freeze-thaw treatment is performed 2 to 4 times.

[0015] Preferably, the heating treatment is carried out in a boiling water bath; The heating time is 5 to 15 minutes; The centrifugal treatment time is 10 to 30 minutes; The rotation speed of the centrifugal treatment is 2000~6000g; The temperature of the centrifugal treatment is 3-5°C.

[0016] Beneficial effects of the present invention: The present invention confirms the excellent application prospects of Dendrobium officinale polypeptide as a cosmetic raw material. It can be seen from the embodiments of the present invention that the present invention selects natural wild Dendrobium officinale with high quality and rich active ingredients as the raw material for extracting plant polypeptides, and measures the molecular weight of the Dendrobium officinale polypeptide to determine that it belongs to a small molecule polypeptide. Through in vitro cell experiments, the application effect of Dendrobium officinale polypeptide in cosmetics was evaluated, and it was proved that Dendrobium officinale polypeptide can promote the expression of collagen-related genes, inhibit the expression of collagen-degrading enzyme-related genes, increase the collagen content, and has an anti-aging and firming effect; inhibit the expression of inflammatory factor-related genes, reduce the occurrence of immune inflammatory reactions, and have an anti-inflammatory effect; inhibit ROS content, reduce oxidative stress, and have an antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the molecular weight distribution diagram of Dendrobium officinale polypeptide; Figure 2 Dendrobium officinale polypeptide regulates collagen COL1A1 Comparison chart of gene expression effects; Figure 3 Dendrobium officinale polypeptide regulates collagen COL3A1 Comparison chart of gene expression effects; Figure 4 Dendrobium officinale polypeptide regulates collagen COL4A1 Comparison chart of gene expression effects; Figure 5 Dendrobium officinale polypeptide regulates matrix metalloproteinases MMP1 Comparison chart of gene expression effects; Figure 6 Dendrobium officinale polypeptide regulates matrix metalloproteinases MMP3 Comparison chart of gene expression effects; Figure 7 Dendrobium officinale polypeptide regulates inflammatory factors IL-1α Comparison chart of gene expression effects; Figure 8 Dendrobium officinale polypeptide regulates inflammatory factors IL-6 Comparison chart of gene expression effects; Fig. 9 Dendrobium officinale polypeptide regulates inflammatory factors IL-8 Comparison chart of gene expression effects; Fig.10 This is a comparison chart of the effect of Dendrobium officinale polypeptides in reducing ROS content. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] The commercially available product information used in the embodiments of the present invention is as follows: Commercially available Vitality Retinol: Merchant: Shanghai Zhina Biotechnology Co., Ltd., trade name: DZ108; Commercially available blue copper peptide: Merchant: Shandong Jipeptide Biotechnology Co., Ltd., trade name: JTBiotech CopperPeptide; Commercially available hydrolyzed collagen: Merchant: DSM, Trade name: SYN ® -COLL; Commercially available Bossagra: Merchant: Shenzhen Readlin Biotechnology Co., Ltd., trade name: Readlin S Bossagra; Commercially available hydrolyzed lupin protein: Merchant information: SILAB, Trade name: STRUCTURINE ® BIO.

[0020] Example 1 Preparation of Dendrobium officinale polypeptide Fresh Dendrobium officinale plants (Dendrobium officinale from Yandang Mountain) were pre-frozen in a -30°C constant temperature refrigerator for 24 h, and then placed in a vacuum freeze dryer for drying. The cold trap temperature was -80°C, the freezing chamber temperature was -45°C-35°C, the vacuum degree was 0-10 Pa, and the vacuum freezing was performed for 48 h. During the 48 h drying process, the freeze-drying procedure used was as follows: Program 1 (1h, -45℃, vacuum 0), Program 2 (0.5h, -45℃, vacuum 0), Program 3 (0.5h, -45℃, vacuum 0-10Pa), Program 4 (0.5h, -30℃, vacuum 0-10Pa), Program 5 (1h, -10℃, vacuum 0-10Pa), Program 6 (5h, 0℃, vacuum 0-10Pa), Program 7 (4h, 5℃, vacuum 0-10Pa), Program 8 ( 3h, 10℃, vacuum degree 0-10Pa), program 9 (5h, 15℃, vacuum degree 0-10Pa), program 10 (5h, 20℃, vacuum degree 0-10Pa), program 11 (5h, 25℃, vacuum degree 0-10Pa), program 12 (5h, 30℃, vacuum degree 0-10Pa), program 13 (5h, 30℃, vacuum degree 0-10Pa), program 14 (7.5h, 35℃, vacuum degree 0-10Pa).

[0021] The mortar and grinding rod were pre-cooled with liquid nitrogen for 3 times, and the freeze-dried Dendrobium officinale was ground into powder, and then placed in a 50 ml centrifuge tube that had been pre-cooled in advance, and added to a 1X PBS solution containing 150 mM NaCl that had been pre-cooled at 4°C in advance at a material-liquid ratio of 1:7. Mixed using a vortex oscillator, repeated freeze-thaw treatment 3 times (freeze in liquid nitrogen, thaw at 37°C), after the treatment, it was placed on ice for 16 min, then boiled in a water bath for 10 min, cooled to room temperature, centrifuged for 20 min (centrifugation conditions 4000 g, 4°C), and the supernatant was taken for purification process.

[0022] Use gel filtration chromatography to purify Dendrobium polypeptides. Before loading the protein sample, it must be filtered through a 0.2 μm filter membrane to remove residues that interfere with chromatography. Balance the chromatography column, and when the equilibrium liquid flows to 1 to 2 mm below the surface of the chromatography bed, close the outlet. Use a sampler to add the Dendrobium polypeptide sample to 2 to 3 cm above the column bed surface, connect the constant pressure sample bottle, and open the outlet to allow the sample to penetrate into the gel.

[0023] After adding the sample, wash the surface 1-2 times with a small volume of eluent, diluting the sample as little as possible. When the sample is close to running dry, slowly and slightly add eluent, so that the eluent reaches 2-5 cm above the surface of the chromatography bed, and then connect the constant pressure elution bottle to start protein purification chromatography. In all the above steps, you must always pay attention to the uniformity of the chromatography surface.

[0024] The solution after chromatography purification is freeze-dried, and the freeze-drying procedure and parameters are the same as above. The freeze-dried powder is the small molecule Dendrobium polypeptide powder.

[0025] Comparative Example 1 Comparative Example 1 uses commercially available active retinol raw material. This raw material has been proven to protect the skin from free radicals; thicken the epidermis and dermis, activate collagen synthesis; participate in the PPAR signaling pathway, and regulate the production of inflammatory factors.

[0026] Comparative Example 2 Comparative Example 2 uses commercially available blue copper peptide raw material, which is known as the ancestor of peptides, one of the three giants of anti-aging. This raw material has been proven to stimulate the production of collagen, inhibit the secretion of inflammatory factors, and protect the skin from free radicals, thereby achieving the effect of improving aging skin and repairing the skin barrier.

[0027] Comparative Example 3 Comparative Example 3 uses commercially available hydrolyzed collagen raw material, which is a product generated by enzymatic hydrolysis of collagen. This raw material has been proven to promote the collagen content of the skin, fight inflammation, free radicals, and reduce skin inflammation and oxidative stress.

[0028] Comparative Example 4 Comparative Example 4 uses commercially available phosphenein raw material. phosphenein, also known as hydroxypropyl tetrahydropyrantriol, has been proven to promote collagen synthesis and tighten skin; it can inhibit inflammation-related biomarkers (IL and TNF-related gene expression), thereby reducing skin inflammatory reactions.

[0029] Comparative Example 5 Comparative Example 5 uses commercially available hydrolyzed lupin protein raw materials and plant active peptides extracted from lupin as raw materials. This raw material has been proven to strengthen type I, type III, and type IV collagen, prevent natural aging; inhibit the activity of matrix metalloproteinases, slow down collagen degradation; reduce ROS content, secretion of inflammatory mediators, and relieve inflammatory reactions caused by oxidative stress.

[0030] Experimental Example 1 Determination of Molecular Weight of Dendrobium officinale Polypeptide 1. Preparation of test molecular weight correction curve Using cytochrome C (standard product), aprotinin (standard product), bacitracin (standard product), tyrosine-tyrosine-arginine (standard product), tyrosine-tyrosine-tyrosine (standard product) as relative molecular weight standards, the molecular weight correction curve was prepared. The specific method is as follows: 1) Weigh an appropriate amount of each standard compound on a 1 / 10,000 balance, place in a 100 mL volumetric flask, use mobile phase (acetonitrile / water / trifluoroacetic acid, volume ratio: 40 / 60 / 0.1) to dissolve and dilute to the mark; 2) After the mixture is dissolved, ultrasonicate it for 5 min and filter it through a 0.22 μm microporous membrane. 3) Place the filtered solution in the GPC sample injection tube and wait for measurement on the machine; 4) Use Empower GPC software to analyze the molecular weight correction curve.

[0031] 2. Mass spectrometry test of Dendrobium officinale polypeptide samples 1) Take 10 mL of Dendrobium officinale polypeptide sample in a 100 mL volumetric flask, dissolve it with mobile phase (acetonitrile / water / trifluoroacetic acid, volume ratio: 40 / 60 / 0.1) and dilute to the mark; 2) After the solution is dissolved, ultrasonicate the sample for 5 min and filter it using a 0.22 μm microporous membrane; 3) Place the filtered solution in the GPC sample injection tube and wait for measurement on the machine; 4) The chromatographic column used in the experiment was Tskgel g2000 SWXL column, column temperature: 30°C; the flow rate of the mobile phase for the test concentration gradient was set at 0.5 ml / min; the sample was detected at an ultraviolet wavelength of 220 nm; 5) Empower GPC software was used to analyze the molecular weight distribution of Dendrobium officinale polypeptide samples. The results are shown in Figure 1 .

[0032] Table 1. Distribution range of molecular weight of Dendrobium officinale polypeptides

[0033] use Figure 1 The mass spectrometry file was used to calculate and analyze the molecular weight distribution of Dendrobium officinale polypeptides using GPC software. The results are shown in Table 1. 97.18% of the molecular weight of the Dendrobium officinale polypeptide samples was distributed between 180 and 3000 Da, and 95.09% of the molecular weight was distributed between 180 and 500 Da. According to the average molecular weight of amino acids of 128 Da, the components in the sample Dendrobium officinale polypeptides are mostly oligopeptides composed of 2 to 5 amino acids.

[0034] Experimental Example 2 Testing of the Firming Effect of Dendrobium polypeptide 1. Expression of collagen-related genes The loss of collagen is considered to be one of the main causes of aging. Collagen mainly maintains the integrity of the fiber structure of the dermis layer of the skin, allowing the skin to absorb water and nutrients more easily. The loss of skin collagen is mainly the loss of type I collagen and type III collagen. The dermis layer of the skin is prone to collapse, resulting in blurred jawline, sagging face, and facial wrinkles.

[0035] In Experimental Example 1, the Dendrobium officinale polypeptide in Example 1, the active retinol in Comparative Example 1, the blue copper peptide in Comparative Example 2, the hydrolyzed collagen in Comparative Example 3, the hydrolyzed lupine protein in Comparative Example 4, and the bosin in Comparative Example 5 were used as test samples, and the method was as follows: 1. Prepare test samples Test example sample 1 (Dendrobium officinale polypeptide): DMEM culture medium containing 10 ppm of Dendrobium officinale polypeptide; Test Example Sample 2 (Active Retinol): DMEM culture medium containing 10 ppm of Active Retinol; Test example sample 3 (blue copper peptide): DMEM culture medium containing 10 ppm blue copper peptide; Test Example Sample 4 (hydrolyzed collagen): DMEM culture medium containing 10 ppm hydrolyzed collagen; Test Example Sample 5 (hydrolyzed lupin protein): DMEM culture medium containing 10 ppm hydrolyzed lupin protein; Test Example Sample 6 (Bosone): DMEM culture medium containing 10 ppm Bosone.

[0036] 2. Preparation of HaCaT:HSF cell co-culture system Take the human immortalized keratinocytes (HaCaT cells) and fibroblasts (HSF cells) in good growth state at a ratio of 1:2, and add 1×10 cells per well. 6 The cells were inoculated at a density of 1000 μg / ml in a 6-well culture plate. 2 Incubate overnight in the environment.

[0037] 3. Modeling After the culture medium was discarded and the cells were washed twice with PBS, the model group and the sample group were given UVB 50 mJ / cm 2 The model was treated with an irradiation dose of .

[0038] 4. Add test samples After modeling, the cells were treated accordingly according to the control group, model group and sample group (sample group: add 2 mL of each component in 1; control group: 2 mL of DMEM culture medium; model group: 2 mL of DMEM culture medium), and each treatment was repeated 3 times at 37°C and 5% CO. 2 Incubate incubator for 24 h.

[0039] 5. RT-qPCR determination to explore the expression of target genes related to firming effect The culture medium of the cells described in 4 was removed, the cells were collected, lysate was added, total RNA of the cells was extracted, and RNA concentration and purity were detected. RT-qPCR technology was used to determine the expression of collagen-related genes COL1A1 (Collagen Type I, type I collagen), COL3A1 (Collagen Type Ⅲ, type Ⅲ collagen), COL4A1 (Collagen Type Ⅳ, type Ⅳ collagen) gene expression level.

[0040] 6. Data Analysis GraphPad Prism 8 software was used, and the data were expressed as mean ± standard error. GraphPad Prism 8 software was used, and the data were expressed as mean ± standard error. The differences between the control group and the model group were analyzed using paired t- test Inspection and analysis, P <0.05 (#) was considered to be significantly different. P <0.01 (##) was considered to be extremely significantly different. P <0.001 (###) was considered to be a highly significant difference; the difference between the sample group and the model group was analyzed using one-way analysis of variance. P <0.05 (*) was considered to be significantly different. P <0.01 (**) was considered to be extremely significantly different.P <0.001 (***) was considered a highly significant difference.

[0041] like Figure 2 , Figure 3 , Figure 4 As shown, compared with the blank control group, after UVB treatment, the cells in the model group COL1A1 , COL3A1 , COL4A1 Gene expression decreased by 52.07% ( P <0.001), 59.9% ( P <0.01), 65.17% ( P <0.01), indicating that the model was successfully established in this experiment and the model group was effective.

[0042] Compared with the model group, the Dendrobium officinale polypeptide significantly increased COL1A1 Gene expression, increasing its expression by 69.59% ( P <0.001). The blue copper peptide had the same significant effect in the control sample group. COL1A1 Gene expression increased by 29.18% ( P <0.001), and other control samples did not significantly promote COL1A1 The effect of expression.

[0043] Compared with the model group, Dendrobium officinale polypeptide can significantly improve COL3A1 , COL4A1 Gene expression, COL3A1 The expression level increased by 137.42% ( P <0.001); COL4A1 The expression level increased by 91.2% ( P <0.001). COL3A1 , COL4A1 The expression increase was not significant ( P >0.05).

[0044] In vitro cell experiments show that the Dendrobium officinale polypeptide of the present invention can promote the expression of collagen-related genes, improve the phenomenon of skin sagging and reduced skin elasticity caused by reduced collagen content, and has a firming effect.

[0045] Experimental Example 2 Detection of the anti-aging effect of Dendrobium officinale polypeptide 1. Collagen Degrading Enzyme-related Genes The degradation of collagen is mainly caused by matrix metalloproteinases (MMPs), which are a highly conserved class of proteases in natural evolution and can degrade almost all components of the extracellular matrix. MMP1 It is the main enzyme that degrades type I and type III collagen. MMP3The hydrolysis substrate of β-actin is relatively broad. It can hydrolyze extracellular matrix proteins such as type III, IV, V, VII, IX and X collagen, elastin, fibronectin, etc.

[0046] In Experimental Example 2, the Dendrobium officinale polypeptide in Example 1, the active retinol in Comparative Example 1, the blue copper peptide in Comparative Example 2, the hydrolyzed collagen in Comparative Example 3, the hydrolyzed lupine protein in Comparative Example 4, and the bosin in Comparative Example 5 were used as test samples, and the method was as follows: 1. Prepare test samples Each sample to be tested was diluted using DMEM culture medium and the samples were marked and grouped.

[0047] Test example sample 1 (Dendrobium officinale polypeptide): DMEM culture medium containing 10 ppm of Dendrobium officinale polypeptide; Test Example Sample 2 (Active Retinol): DMEM culture medium containing 10 ppm of Active Retinol; Test example sample 3 (blue copper peptide): DMEM culture medium containing 10 ppm blue copper peptide; Test Example Sample 4 (hydrolyzed collagen): DMEM culture medium containing 10 ppm hydrolyzed collagen; Test Example Sample 5 (hydrolyzed lupin protein): DMEM culture medium containing 10 ppm hydrolyzed lupin protein; Test Example Sample 6 (Bosone): DMEM culture medium containing 10 ppm Bosone.

[0048] 2. Preparation of HaCaT:HSF cell co-culture system Same as Experimental Example 1.

[0049] 3. Modeling Same as Experimental Example 1.

[0050] 4. Add test samples Same as Experimental Example 1.

[0051] 5. RT-qPCR determination of cell barrier-related target gene expression The culture medium of the cells described in 4 was removed, the cells were collected, and lysis buffer was added to extract the total RNA of the cells, and the RNA concentration and purity were detected. RT-qPCR technology was used to determine the collagen degrading enzyme-related genes MMP1 (Matrix Metallopeptidase 1, matrix metalloproteinase 1), MMP3 (Matrix Metallopeptidase 3, matrix metalloproteinase 3) gene expression level.

[0052] 6. Data processing Same as Experimental Example 1.

[0053] like Figure 5 , Figure 6 As shown, compared with the blank control group, the cells in the model group were treated with UVB. MMP1 Gene expression increased by 90.15% ( P <0.001), indicating that the model group was effective; compared with the model group, Dendrobium officinale polypeptide could inhibit MMP1 gene expression, causing its expression to decrease by 27.51% ( P <0.05); in the control sample group, the blue copper peptide still had the best effect. MMP1 The inhibition rate of gene expression was 26.18% ( P <0.05).

[0054] like Figure 5 As shown, compared with the blank control group, the cells in the model group were treated with UVB. MMP3 Gene expression increased by 121.64% ( P <0.001), indicating that the model group was effective; compared with the model group, Dendrobium officinale polypeptide could inhibit MMP3 gene expression, causing its expression to decrease by 16.54% ( P <0.001); control sample group MMP3 The inhibitory effects on gene expression were not significant ( P >0.05).

[0055] The results of in vitro cell experiments show that Dendrobium officinale polypeptide can inhibit the expression of matrix metalloproteinase genes, inhibit collagen degradation, maintain skin structure, and improve the skin's anti-aging ability.

[0056] Experimental Example 3 Detection of the anti-inflammatory effect of Dendrobium officinale polypeptide IL-1α, IL-6 and IL-8 All are pro-inflammatory cytokines. They are biologically active both inside and outside cells. As an inflammatory mediator, IL-1α, IL-6 and IL-8 It can trigger an inflammatory response, promote the activation of immune cells and the release of other inflammatory mediators.

[0057] In Experimental Example 3, the Dendrobium officinale polypeptide in Example 1, the active retinol in Comparative Example 1, the blue copper peptide in Comparative Example 2, the hydrolyzed collagen in Comparative Example 3, the hydrolyzed lupine protein in Comparative Example 4, and the bosin in Comparative Example 5 were used as test samples, and the method was as follows: 1. Prepare test samples Each sample to be tested was diluted using DMEM culture medium and the samples were marked and grouped.

[0058] Test example sample 1 (Dendrobium officinale polypeptide): DMEM culture medium containing 10 ppm of Dendrobium officinale polypeptide; Test Example Sample 2 (Active Retinol): DMEM culture medium containing 10 ppm of Active Retinol; Test example sample 3 (blue copper peptide): DMEM culture medium containing 10 ppm blue copper peptide; Test Example Sample 4 (hydrolyzed collagen): DMEM culture medium containing 10 ppm hydrolyzed collagen; Test Example Sample 5 (hydrolyzed lupin protein): DMEM culture medium containing 10 ppm hydrolyzed lupin protein; Test Example Sample 6 (Bosone): DMEM culture medium containing 10 ppm Bosone.

[0059] 2. Preparation of HaCaT:HSF cell co-culture system Same as Experimental Example 1.

[0060] 3. Modeling Same as Experimental Example 1.

[0061] 4. Add test samples Same as Experimental Example 1.

[0062] 5. RT-qPCR determination of cell barrier-related target gene expression The culture medium of the cells described in 3 was removed, the cells were collected, and lysis buffer was added to extract the total RNA of the cells, and the RNA concentration and purity were detected. RT-qPCR technology was used to determine the genes related to inflammatory factors IL-1 (Interleukin 1α, interleukin 1α), IL- 6 (Interleukin 6, interleukin 6), IL-8 (Interleukin 8, interleukin 8) gene expression level.

[0063] 6. Data Analysis Same as Experimental Example 1.

[0064] like Figure 7 , 8 As shown in Figure 9, compared with the blank control group, the three classic inflammatory factors ( IL-1α, IL-6 and IL-8 ) gene expression levels were significantly increased by 36.72% ( P <0.05) and 261.36% ( P <0.001) and 299.05% ( P <0.001), indicating that the model was successfully established in this experiment and the model group was effective.

[0065] Compared with the model group, the sample Dendrobium officinale polypeptide can simultaneously inhibit the expression of these three inflammatory factor genes. IL-1α, IL-6 and IL-8 Gene expression decreased by 17.52% ( P <0.05), 57.06% ( P <0.001) and 39.85% ( P <0.05).

[0066] Inflammatory factors in the control group IL-1α and IL-8 The gene expression inhibition effect was not significant ( P >0.05). The control group samples showed no significant difference in inflammatory factors IL-1α and IL-8 The gene expression inhibition effect is significant, and blue copper peptide can inhibit IL-6 gene expression, causing its expression to decrease by 42.94% ( P <0.001).

[0067] The results of in vitro cell experiments showed that Dendrobium officinale polypeptide can inhibit IL-1α, IL-6 and IL-8 The gene expression of inflammatory factors can reduce the immune inflammatory response caused by inflammatory factors and have an anti-inflammatory effect.

[0068] Experimental Example 4 Detection of the Antioxidant Effect of Dendrobium polypeptide In Experimental Example 4, the Dendrobium officinale polypeptide in Example 1, the active retinol in Comparative Example 1, the blue copper peptide in Comparative Example 2, the hydrolyzed collagen in Comparative Example 3, the hydrolyzed lupine protein in Comparative Example 4, and the bosin in Comparative Example 5 were used as test samples, and the method was as follows: 1. Prepare test samples Test example sample 1 (Dendrobium officinale polypeptide): DMEM culture medium containing 10 ppm of Dendrobium officinale polypeptide; Test Example Sample 2 (Active Retinol): DMEM culture medium containing 10 ppm of Active Retinol; Test example sample 3 (blue copper peptide): DMEM culture medium containing 10 ppm blue copper peptide; Test Example Sample 4 (hydrolyzed collagen): DMEM culture medium containing 10 ppm hydrolyzed collagen; Test Example Sample 5 (hydrolyzed lupin protein): DMEM culture medium containing 10 ppm hydrolyzed lupin protein; Test Example Sample 6 (Bosone): DMEM culture medium containing 10 ppm Bosone.

[0069] 2. Preparation of HaCaT:HSF cell co-culture system Same as Experimental Example 1.

[0070] 3. Modeling Same as Experimental Example 1.

[0071] 4. Add test samples Same as Experimental Example 1.

[0072] 5. Determination of ROS content by DCFH-DA method Discard the 4 culture medium, wash twice with PBS and collect the cells. Dilute DCFH-DA with serum-free culture medium at 1:500 to a final concentration of 10 μmol / L. After collecting the cells, suspend them in the diluted DCFH-DA at a cell concentration of 1 million to 20 million / ml and incubate in a 37°C cell culture incubator for 20 minutes. Invert and mix every 3-5 minutes to allow the probe and cells to fully contact. Wash the cells once with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. After collecting the cells, use a fluorescent microplate reader to detect at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0073] 6. Data Analysis Same as Experimental Example 1.

[0074] like Fig.10 As shown in the figure, compared with the blank control group, the reactive oxygen content in the model group increased significantly by 62.47% after UVB treatment ( P <0.01), indicating that the model group is effective.

[0075] Compared with the model group, the Dendrobium officinale polypeptide could significantly inhibit the release of reactive oxygen species, reducing it by 37.39% ( P <0.001); the inhibition rate of active retinol on reactive oxygen species in the control group was 39.27% ​​( P <0.001); the inhibition rate of blue copper peptide on reactive oxygen species was 30.03% ( P <0.001); the inhibition rate of hydrolyzed collagen on active oxygen was 31.01% ( P <0.001); the inhibition rate of hydrolyzed lupin protein on active oxygen was 26.89% ( P <0.01); the inhibition rate of Bossagra on active oxygen was 27.65% ( P <0.001).

[0076] The above experimental results show that Dendrobium officinale polypeptide can inhibit the content of active oxygen, improve the skin's ability to cope with oxidative stress, and has an antioxidant effect.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of a Dendrobium officinale polypeptide in the preparation of cosmetics.

2. The use according to claim 1, characterized in that: The cosmetic is a skin anti-aging product.

3. The use according to claim 1, characterized in that: The cosmetic is an anti-inflammatory product.

4. The use according to claim 1, characterized in that: The cosmetic is an antioxidant product.

5. The use according to any one of claims 1 to 4, characterized in that: The Dendrobium officinale polypeptide is obtained by a preparation method comprising the following steps: freeze-drying and pulverizing the Dendrobium officinale in sequence to obtain Dendrobium officinale powder; The powder of Dendrobium officinale is mixed with a buffer solution containing NaCl, and subjected to freeze-thaw treatment to obtain an intermediate; The intermediate is placed at -5 to 5°C for 5 to 20 minutes, and then subjected to heating treatment and centrifugation treatment in sequence to obtain a supernatant; The supernatant is freeze-dried to obtain the Dendrobium officinale polypeptide.

6. The use according to claim 5, characterized in that: The Dendrobium officinale is pre-frozen before freeze-drying.

7. The use according to claim 6, characterized in that: The pre-freezing temperature is -40 to -20°C; The pre-freezing time is 12 to 36 hours.

8. The use according to claim 6, characterized in that: The temperature of the freeze-drying cold trap is -100~-60°C; The freezer temperature of the freeze-drying chamber is -45°C to 35°C; The vacuum degree of the freeze drying is 0-10 Pa; The freeze-drying time is 36 to 72 hours.

9. The use according to claim 1, characterized in that: The solid-liquid ratio of the Dendrobium officinale powder and the buffer solution containing NaCl is 1:5-10; The buffer is PBS; The final concentration of NaCl in the buffer is 100-200 mM; The freezing stage in the freeze-thaw treatment adopts liquid nitrogen freezing; The temperature used in the thawing stage of the freeze-thaw treatment is 35-40°C; The freeze-thaw treatment is performed 2 to 4 times.

10. The use according to claim 1, characterized in that: The heating treatment is carried out in a boiling water bath; The heating time is 5 to 15 minutes; The centrifugal treatment time is 10 to 30 minutes; The rotation speed of the centrifugal treatment is 2000~6000g; The temperature of the centrifugal treatment is 3-5°C.

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