Arabidopsis lazy mutant extract, preparation method thereof, and application in anti-skin aging

Abstract

The present invention belongs to the field of anti-skin aging, and specifically relates to an Arabidopsis lazy The mutant extract and its preparation method and application in anti-skin aging. The preparation method comprises the steps of washing the plant, gradient drying, crushing, ethanol ultrasonic extraction, rotary evaporation concentration, etc. lazy Mutant extracts were upregulated by TERT gene expression (+52.58%), enhanced mitochondrial activity (ATP+19.77%), promoted collagen synthesis ( COL1A1 +196.89%, COL3A1 +243.81%, ELN The invention significantly delays skin aging through multiple pathways, including reducing skin aging by 187.04% (+187.04%) and increasing cell survival by 76.47%. The process is simple and efficient, making it suitable for the development of anti-aging cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of anti-skin aging, and specifically relates to an Arabidopsis lazy Mutant extract, preparation method thereof and application in anti-skin aging. Background Art

[0002] As we age, our skin develops wrinkles, sagging, and spots, among other aging issues. Modern scientific research indicates that numerous factors influence skin aging, resulting from a combination of both external and internal factors. External factors primarily include poor diet, lack of sleep, stress, UV exposure, and air pollution, while internal factors can be summarized as genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, loss of macrophage function, nutrient sensing disorders, mitochondrial dysfunction, cellular senescence, stem cell failure, altered intercellular communication, chronic inflammation, and dysbiosis. Therefore, the development of cosmetic ingredients with anti-aging properties is of great significance in the cosmetics industry.

[0003] Gravity, as a physical factor that exists everywhere on Earth at all times, plays an extremely important regulatory role in the growth and development of plants, but it also has adverse effects on human skin and body aging. The roots of plants grow toward the center of the earth (positive gravity), which is beneficial for the plant's fixation and absorption of water and nutrients in the soil; while the stems of plants grow upward (negative gravity), which is beneficial for obtaining better lighting conditions and growth space. The most widely accepted theory of plant gravity perception is the "starch-stability stone" hypothesis. LAZY protein plays a key role in the process of plant roots sensing gravity. Arabidopsis thaliana lazy The mutant exhibits gravity insensitivity, with the root tip exhibiting a phenotype of randomly oriented growth. The molecular mechanism underlying this is summarized as follows: when plants grow vertically, the starch spherical cells in the root apex settle to the underside of the cells, where LAZY proteins are primarily localized to the cell membrane and exhibit low phosphorylation levels. When plants deviate from gravity, the interaction between the MKK5-MPK3 protein kinase module and LAZY proteins increases, boosting their phosphorylation. This phosphorylated LAZY protein interacts more strongly with TOC34, TOC120, and TOC132 proteins on the starch spherical surface, promoting its translocation from the cell membrane to the outer starch spherical membrane. The settling of the starch spherical cells directs the LAZY proteins to accumulate on the new underside of the stylus cells, where they regulate the asymmetric distribution of auxin and the curvilinear growth of the root. When the root apex returns to its vertical orientation, the phosphorylation and localization of the LAZY proteins return to their initial state. LAZY protein is a key protein for plants to sense gravity. After the LAZY protein in Arabidopsis thaliana mutates, the mutant cannot sense gravity, and the roots do not have a downward (geocentric) growth phenotype. However, at this stage, there is no research linking LAZY protein with anti-aging.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a lazy A method for preparing a mutant extract, comprising the following steps:

[0006] (1) Collect unbolted Arabidopsis lazy The complete mutant plant was washed and dried to obtain dried Arabidopsis thaliana lazy mutants;

[0007] (2) drying the Arabidopsis thaliana lazy The mutant is crushed, mixed with a solvent, and then subjected to ultrasonic extraction and filtration to obtain an extract;

[0008] (3) The extract is rotary evaporated to remove the solvent, thereby obtaining the Arabidopsis thaliana lazy Mutant extracts.

[0009] Preferably, in step (1), the number of cleanings is 2 to 3 times.

[0010] Preferably, in step (1), the drying method is: first drying at 100-105°C for 15-20 minutes, and then drying at 60-65°C for 4-5 hours.

[0011] Preferably, in step (2), the average particle size of the crushed product is 200-250 mesh.

[0012] Preferably, in step (2):

[0013] The solvent is ethanol;

[0014] And / or, the drying of Arabidopsis thaliana lazy The mass ratio of mutant to solvent was 1:10–12;

[0015] And / or, the ultrasonic extraction method is: extraction at 25-45° C. and an ultrasonic power of 2000-2200 W for 0.5-1 h.

[0016] Preferably, in step (3), the rotary evaporation is carried out as follows: first, rotary evaporation is carried out at 35-42°C and 80-120 r / min until the liquid stops overflowing, and then the rotary evaporation temperature is increased to 48-52°C, and rotary evaporation is carried out for 30-40 minutes until the solvent is completely removed.

[0017] Based on the same technical concept, another embodiment of the present invention is to provide an Arabidopsis thaliana obtained by the preparation method. lazy Mutant extracts.

[0018] Based on the same technical concept, another embodiment of the present invention is to provide an Arabidopsis thaliana lazy Application of mutant extracts in the preparation of anti-skin aging products.

[0019] Preferably, the anti-skin aging product is a product that protects telomeres;

[0020] And / or, the anti-skin aging product is a product that enhances mitochondrial activity;

[0021] and / or, the anti-skin aging product is a product that increases the collagen content of ECM fibers;

[0022] And / or, the anti-skin aging product is an anti-cell aging product.

[0023] The beneficial effects of the present invention are:

[0024] 1. The preparation method of the present invention is to harvest Arabidopsis thaliana lazy The mutant whole plant was finally obtained through washing, drying, extraction, rotary evaporation and other steps. lazy The mutant extract has a simple and easy-to-control overall process, which is conducive to large-scale production.

[0025] 2. After detailed experiments, it was confirmed that the Arabidopsis thaliana obtained by the present invention lazy The mutant extract has the effect of protecting and delaying cell aging by protecting telomeres, improving mitochondrial activity, increasing collagen expression and collagen fiber quantity, and resisting cell apoptosis. It can make outstanding contributions in the field of anti-skin aging, specifically:

[0026] (2-1) Telomere protection: The extract significantly improves TERT Gene expression (52.58%, p<0.05 ), delaying cell replicative senescence;

[0027] (2-2) Mitochondrial activation: Under UVB damage, the mitochondrial fluorescence intensity increased by 36.40% ( p<0.01 ), ATP content increased by 19.77% ( p<0.01 ), effectively maintain cellular energy metabolism;

[0028] (2-3) Collagen promotion: COL1A1 、 COL3A1 and ELN The gene expression levels increased by 196.89%, 243.81% and 187.04% respectively ( p<0.001 ), collagen fiber content increased by 149.26% ( p<0.001 ), significantly improve skin elasticity and firmness;

[0029] (2-4) Anti-cell aging: The proportion of β-galactosidase positive cells decreased by 76.47% ( p<0.001 ), which was confirmed to be a potent inhibitor of photoaging-induced cellular senescence.

[0030] (2-5) Wide application: The extract can be used in a variety of anti-skin aging products such as protecting telomeres, enhancing mitochondrial activity, promoting collagen synthesis and anti-cellular aging, providing innovative raw materials for the cosmetics and medical beauty fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Arabidopsis thaliana lazy Schematic diagram of the mutant DNA sequence deletion.

[0033] Figure 2 Telomerase reverse transcriptase gene ( TERT ) Gene expression analysis data graph.

[0034] Figure 3 This is a cell mitochondrial activity analysis graph, including the mitochondrial relative fluorescence intensity graph (left) and the ATP content graph in the cell (right).

[0035] Figure 4 It's collagen COL1A1 Gene expression analysis data graph.

[0036] Figure 5 It's collagen COL3A1 Gene expression analysis data graph.

[0037] Figure 6 It is elastin ELN Gene expression analysis data graph.

[0038] Figure 7 This is an analysis chart of the collagen fiber content in cells, including a collagen fiber staining (blue) chart under a microscope (left) and a statistical chart of the collagen fiber content ratio (right).

[0039] Figure 8 This is a cell aging analysis chart, including a microscopic image of β-galactosidase-positive cell staining (blue) (left) and a statistical chart of the proportion of β-galactosidase-positive cells (right). DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] This example first provides a stable Arabidopsis lazy A method for obtaining a mutant, comprising the following steps:

[0043] (1) Vector modification: The rbcS-E9 terminator on the pHEE401E vector was replaced with the Nos terminator on the p35S-Cas9-SK vector by the restriction sites of BamHⅠ and EcoRⅠ to form the p35S-Cas9-rbcS-E9t vector; the restriction site XhoⅠ upstream of the double CaMV 35S promoter on the p35S-Cas9-rbcS-E9t vector was deleted, and the restriction site NheⅠ was inserted between the restriction sites SalesⅠ and HindⅢ to form the p35S-Cas9-rbcS-E9t-M vector; the promoter Ec1.2enEc1.1 of pHEE401E was replaced with the double CaMV 35S promoter on the p35S-Cas9-rbcS-E9t-M vector by the restriction sites of NheⅠ and XhoⅠ to finally form the pEc1.2enEc1.1p-Cas9-rbcS-E9t vector.

[0044] (2) Vector construction: Two sgRNAs were designed for LAZY gene family members LAZY2, LAZY3, and LAZY4, respectively. The primers are shown in Table 1. The six designed sgRNAs were constructed into the intermediate vector pAtU6-26-M with the restriction site BbsⅠ. The expression cassette containing the six sgRNAs was constructed into the pEc1.2enEc1.1p-Cas9-rbcS-E9t vector with the restriction sites KpnⅠ and SalⅠ. Finally, the Cas9 expression cassette (containing the six sgRNAs) was constructed into the pCAMBIA1300 vector (hygromycin resistance) with the restriction sites KpnⅠ and EcoRⅠ, finally forming the pCAMBIA1300-LAZY2 / 3 / 4 sgRNA vector.

[0045] (3) Genetic transformation: The above-mentioned vector pCAMBIA1300-LAZY2 / 3 / 4 sgRNA was transferred into GV3101 Agrobacterium, and transgenic offspring were obtained by Agrobacterium-inflorescence infection. Positive plants were obtained by hygromycin selection marker and DNA sequence identification. After separation and screening of the offspring, Arabidopsis thaliana without the pCAMBIA1300-LAZY2 / 3 / 4 sgRNA vector and stable inheritance were finally obtained. lazy mutant.

[0046] According to the DNA sequencing results of the target genes, it was confirmed that the DNA sequences between the two sgRNAs of the LAZY2, LAZY3 and LAZY4 genes were deleted or inverted, which resulted in the inability to express the genes normally, thus forming mutants. Specifically, the LAZY2 gene lost 448bp, the LAZY3 gene lost 224bp, and the LAZY4 gene first lost 17bp, then had a 343bp DNA sequence inversion and finally lost 2bp. The mutation sites are shown in Figure 1 .

[0047] Table 1 Primer sequences used to construct lazy mutants in Arabidopsis

[0048]

[0049] Example 2

[0050] This embodiment provides a kind of Arabidopsis lazy A method for preparing a mutant extract, comprising the following steps:

[0051] (1) Harvest healthy, unbolted Arabidopsis lazy The complete mutant plant (including above-ground and underground parts) was rinsed twice with running water and then drained, and then dried in a 105°C oven for 20 min, and then transferred to a 65°C oven for continuous drying for 5 hours to obtain dried Arabidopsis thaliana. lazy mutants;

[0052] (2) Accurately weigh 500g of the dried Arabidopsis thaliana lazy The mutant was ground into a fine powder (average particle size of approximately 200 mesh) using a grinder. 5 L of 50% ethanol solution was added at a solid-liquid ratio (m / m) of 1:10. The mixture was extracted at 25°C and an ultrasonic power of 2200 W for 0.5 h. The extract was filtered through a Buchner funnel and a 0.22 μm filter membrane to obtain an extract. The ultrasonic and Buchner funnel and 0.22 μm filter membrane steps were repeated three times to obtain an extract.

[0053] (3) The extract was placed in a rotary evaporator and evaporated at 35°C and 120 r / min until the liquid did not overflow. The temperature was then raised to 48°C and the evaporation was continued for 40 min until the ethanol was completely removed. Water was added to make the volume 5 L to obtain Arabidopsis thaliana. lazy Mutant extracts.

[0054] Example 3

[0055] This embodiment provides a kind of Arabidopsis lazy A method for preparing a mutant extract, comprising the following steps:

[0056] (1) Harvest healthy, unbolted Arabidopsis lazy The complete mutant plant (including above-ground and underground parts) was rinsed with running water three times and then drained, and then dried in a 100°C oven for 15 minutes, and then transferred to a 60°C oven for continuous drying for 4 hours to obtain dried Arabidopsis thaliana. lazy mutants;

[0057] (2) Accurately weigh 500g of the dried Arabidopsis thaliana lazy The mutant was ground into a fine powder (average particle size of approximately 250 mesh) using a grinder. 5 L of 50% ethanol solution was added at a solid-liquid ratio (m / m) of 1:10. The mixture was extracted at 45°C and an ultrasonic power of 2000 W for 1 h. The extract was filtered through a Buchner funnel and a 0.22 μm filter membrane to obtain an extract. The ultrasonic and Buchner funnel and 0.22 μm filter membrane steps were repeated three times to obtain an extract.

[0058] (3) The extract was placed in a rotary evaporator and evaporated at 42°C and 80 r / min until the liquid did not overflow. Then the rotary evaporation temperature was increased to 52°C and the evaporation was continued for 30 min until the ethanol was completely removed. Water was added to make the volume 5 L to obtain Arabidopsis thaliana. ​ Mutant extracts.

[0059] Example 4

[0060] This embodiment provides a kind of Arabidopsis ​ A method for preparing a mutant extract, comprising the following steps:

[0061] (1) Harvest healthy, unbolted Arabidopsis ​ The complete mutant plant (including above-ground and underground parts) was rinsed with running water three times and then drained, and then dried in a 102°C oven for 18 minutes, and then transferred to a 62°C oven for continuous drying for 4 hours to obtain dried Arabidopsis thaliana. ​ mutants;

[0062] (2) Accurately weigh 500g of the dried Arabidopsis thaliana ​The mutant was ground into a fine powder (average particle size of approximately 220 mesh) using a grinder. 5 L of 50% ethanol solution was added at a solid-liquid ratio (m / m) of 1:10. The mixture was extracted at 35°C and an ultrasonic power of 2100 W for 0.8 h. The extract was filtered through a Buchner funnel and a 0.22 μm filter membrane to obtain an extract. The ultrasonic and Buchner funnel and 0.22 μm filter membrane steps were repeated three times to obtain an extract.

[0063] (3) The extract was placed in a rotary evaporator and first evaporated at 40°C and 100 r / min until the liquid did not overflow. Then the rotary evaporation temperature was increased to 50°C and the evaporation was continued for 35 minutes until the ethanol was completely removed. Water was added to make the volume 5 L to obtain Arabidopsis thaliana. ​ Mutant extracts.

[0064] Verification Example 1: Arabidopsis ​ Evaluation of telomere-protective efficacy of mutant extracts

[0065] Telomeres are repetitive DNA sequences at the ends of eukaryotic chromosomes. The length of telomeres reflects the replication history and replication potential of cells and is known as the "mitotic clock" of cell life. ​ ​ , ​ ) plays an important role in maintaining telomere stability and cell aging. Therefore, maintaining and improving ​ Gene expression is of great significance for maintaining cell vitality and fighting aging.

[0066] The experimental group was divided into control group, model group, experimental group 1 (wild-type Arabidopsis thaliana extract) and experimental group 2 (Arabidopsis thaliana extract). ​ Mutant extracts), three biological replicates were set for each group. 1×10 HaCaT cells in the logarithmic growth phase were taken 6 Cells were seeded into 6-well plates, 2 mL of DMEM medium was added, and cultured at 37°C, 5% CO2 for 24 hours. The culture medium was discarded, and 2 mL of fresh DMEM medium was added. Braco-19 was added to a final concentration of 10 μM in the model group, Braco-19 was added to a final concentration of 10 μM and wild-type Arabidopsis extract was added to a final concentration of 0.5% in experimental group 1, and Braco-19 was added to a final concentration of 10 μM and Arabidopsis extract was added to a final concentration of 0.5% in experimental group 2. ​ The mutant extract was raised to a final concentration of 0.5%. The cells were cultured at 37°C, 5% CO2 for 24 h. The culture medium was discarded, the cells were washed twice with 0.1 M PBS, and the cells were collected by centrifugation at 3000 rpm for 5 min. RNA was extracted, reverse transcribed into cDNA, and analyzed by RT-qPCR. ​ Gene expression level.

[0067] ​It shows that the model group treated with Braco-19 is better than the control group without treatment. ​ The gene expression level was significantly downregulated, indicating that the experimental model was successful and can be used to detect Arabidopsis ​ Protective effect of mutant extracts on telomeres; Adding Arabidopsis thaliana under the premise of adding Braco-19 ​ Mutant extracts in experimental group 2 ​ The gene expression level increased significantly compared with the model group, with an increase of 52.58% ( p<0.05 ); In experimental group 1, wild-type Arabidopsis extract was added under the premise of adding Braco-19. ​ The gene expression level also showed a slight increase compared with the model group, with an increase of 14.67% (no significance), indicating that Arabidopsis ​ The mutant extracts showed significant telomere protection effects.

[0068] Verification Example 2: Arabidopsis ​ Evaluation of the efficacy of mutant extracts in improving mitochondrial activity

[0069] Mitochondria are the "energy factories" of cells, and their functional state is closely related to aging. Changes such as decreased mitochondrial activity, DNA mutations, excessive production of free radicals (ROS), and decreased energy metabolism can drive cellular aging by disrupting cellular homeostasis, triggering chronic inflammation, and accelerating cell damage. Therefore, maintaining or enhancing mitochondrial activity is beneficial for maintaining cell health and vitality, slowing the aging process.

[0070] The experimental group was divided into control group, model group, experimental group 1 (wild-type Arabidopsis thaliana extract) and experimental group 2 (Arabidopsis thaliana extract). ​ Mutant extracts), three biological replicates were set for each group. 1×10 HaCaT cells in the logarithmic growth phase were taken 6 Cells were seeded into 6-well plates, 2 mL of DMEM medium was added, and cultured at 37°C, 5% CO2 for 24 h. The culture medium was discarded, and wild-type Arabidopsis extract was added to a final concentration of 0.5% in experimental group 1, and Arabidopsis extract was added to experimental group 2. ​ The mutant extract was added to a final concentration of 0.5%, and the control group and model group were added with the same amount of serum-free medium. The cells in the model group, experimental group 1 and experimental group 2 were treated with 60 J / cm 2 Treat with UVB and incubate at 37°C, 5% CO₂ for 24 hours. Add Mito-Tracker Green dye to a final concentration of 0.1 µM and incubate at 37°C for 30 minutes. Discard the culture medium and add preheated culture medium at 37°C. Observe fluorescence intensity under a fluorescence microscope to determine mitochondrial activity. Simultaneously, use an enhanced ATP assay kit to measure cellular ATP content.

[0071] ​ Shows: 60 J / cm 2 The UVB-irradiated model group showed a significant decrease in mitochondrial fluorescence intensity and ATP content in cells compared to the untreated control group, indicating that the model was successfully established and can be used to detect Arabidopsis thaliana. ​ Effects of mutant extracts on mitochondrial activity; at 60 J / cm 2 Arabidopsis thaliana ​ The mutant extract significantly enhanced the fluorescence intensity of mitochondria in cells compared with the model group, increasing by 36.40% ( p<0.01 ), ATP content increased significantly, increasing by 19.77% ( p<0.01 ); The addition of wild-type Arabidopsis extract slightly increased the fluorescence intensity of mitochondria in cells compared with the model group, increasing by 9.06% (not significant), and the ATP content slightly increased by 5.25% (not significant). In summary, Arabidopsis ​ The mutant extract has the effect of significantly enhancing mitochondrial activity after cells are damaged by UVB.

[0072] Verification Example 3: Arabidopsis ​ Evaluation of the efficacy of mutant extracts in increasing collagen content in ECM fibers

[0073] As we age, collagen synthesis in skin cells decreases and loss accelerates. In addition, the stimulation of the external environment, especially ultraviolet rays, will destroy the elastic fibers and collagen in the dermis of the skin, accelerating collagen degradation and loss, and ultimately leading to skin sagging and aging. Therefore, increasing collagen expression plays an important role in maintaining skin firmness.

[0074] The experimental group was divided into control group, model group, experimental group 1 (wild-type Arabidopsis thaliana extract) and experimental group 2 (Arabidopsis thaliana extract). ​ Mutant extracts), three biological replicates were set for each group. 1×10 HSP cells in the logarithmic growth phase were collected. 6 Cells were seeded into 6-well plates, 2 mL of DMEM medium was added, and cultured at 37°C, 5% CO2 for 24 h. The culture medium was discarded, and wild-type Arabidopsis extract was added to a final concentration of 0.5% in experimental group 1, and Arabidopsis extract was added to experimental group 2. ​ The mutant extract was added to a final concentration of 0.5%, and the control group and model group were added with the same amount of serum-free medium. The cells in the model group, experimental group 1 and experimental group 2 were treated with 60 J / cm 2 Treat with UVB and culture at 37°C, 5% CO2 for 24 h.

[0075] 1) Collagen gene ​ and ​ , elastin gene ​Expression analysis: The supernatant was discarded, the cells were collected, and total RNA was extracted using an RNA extraction kit. The RNA was reverse transcribed into cDNA, and RT-qPCR was performed to determine the gene expression level.

[0076] 2) Collagen Fiber Content Assay: Total collagen fiber content was determined using a Masson's trichrome staining kit. (Discard the supernatant, wash twice with PBS, and fix with 1 mL of 4% tissue fixative at room temperature for 20 min. Discard the supernatant, stain with 1 mL of mordant at 58°C for 1 h, rinse with running water for 10 min, stain with lapis lazuli blue solution for 2 min, rinse twice with distilled water for 15 s each, differentiate with acidic differentiation solution for 20 s, terminate differentiation by washing with water, and rinse with distilled water for 10 min. Stain with Ponceau fuchsin solution for 10 min, rinse twice with distilled water for 15 s each, treat with concentrated phosphomolybdic acid solution for 10 min, discard the supernatant, and stain with aniline blue solution for 5 min. Remove the aniline blue solution by washing with weak acid solution. Dehydrate with 95% ethanol for 30 s and then with 100% ethanol for 30 s. After staining, collagen fibers appear blue. Cell images were acquired using a fluorescence microscope, and collagen fiber content was analyzed using Image J software.)

[0077] ​ 、 ​ 、 ​ 、 ​ Shows: 60 J / cm 2 The UVB-irradiated model group was compared with the untreated control group. ​ 、 ​ and ​ The relative expression of genes was significantly downregulated and the proportion of collagen fiber content decreased significantly, indicating that the experimental model was successful and can be used to detect Arabidopsis thaliana. ​ Effects of mutant extracts on total ECM collagen; Addition of Arabidopsis ​ In the experimental group of mutant extracts ​ 、 ​ and ​ The relative expression of genes was significantly increased compared with the model group, reaching a very significant level, increasing by 196.89% ( p<0.001 )、243.81%( p<0.001 ) and 187.03% ( p< 0.001 ), while the collagen fiber content increased significantly, increasing by 149.26% ( p<0.001 ); while in the experimental group with wild-type Arabidopsis extract ​ 、 ​ and ​ The relative expression of genes was significantly increased compared with the model group, increasing by 82.76% ( p<0.05 )、105.98%( p<0.01 ) and 87.10% ( p<0.001), the collagen fiber content increased significantly, increasing by 55.56% ( p<0.05 ). Add Arabidopsis ​ Compared with the experimental group 1 with wild-type Arabidopsis extract, the experimental group 2 with mutant extract showed a significant difference in the above ​ 、 ​ 、 ​ The relative expression of the gene and the percentage of collagen fiber content showed a more significant increase, indicating that it has a better effect in increasing the collagen fiber content. ​ The mutant extract significantly increased collagen gene expression after cells were damaged by UVB ​ 、 ​ and elastin genes ​ expression and increase collagen fiber content.

[0078] Verification Example 4: Arabidopsis ​ Evaluation of the anti-aging efficacy of mutant extracts

[0079] Long-term exposure to UV rays can cause a variety of skin problems, including photoaging, hyperpigmentation, dermatitis, sunburn, and even skin cancer. UV rays can accelerate cell aging and even cell death, leading to skin sagging and aging. Therefore, finding suitable cosmetic ingredients to address UV-induced skin aging is crucial for developing novel anti-photoaging cosmetics.

[0080] The experimental group was divided into control group, model group, experimental group 1 (wild-type Arabidopsis thaliana extract) and experimental group 2 (Arabidopsis thaliana extract). ​ Mutant extracts), three biological replicates were set for each group. 1×10 HSP cells in the logarithmic growth phase were collected. 6 Cells were seeded into 6-well plates, 2 mL of DMEM medium was added, and cultured at 37°C, 5% CO2 for 24 h. The culture medium was discarded, and wild-type Arabidopsis extract was added to a final concentration of 0.5% in experimental group 1, and Arabidopsis extract was added to experimental group 2. ​ The mutant extract was added to a final concentration of 0.5%, and the control group and model group were added with the same amount of serum-free medium. The cells in the model group, experimental group 1 and experimental group 2 were treated with 60 J / cm 2Treat with UVB and incubate at 37°C, 5% CO2 for 24 hours. Discard the supernatant, wash twice with PBS for 3 minutes each time, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 minutes. Discard the supernatant, wash three times with PBS for 3 minutes each time, add 1 mL of staining solution, and capture cell images under a microscope. Image J software is used to analyze the proportion of β-galactosidase-positive cells (indicating senescent cells, displayed in blue) in the images. The relative content is calculated as follows: β-galactosidase-positive cell relative content (%) = (proportion of β-galactosidase-positive cells in the model group or experimental group / proportion of β-galactosidase-positive cells in the blank control group) * 100%

[0081] ​ Shows: 60 J / cm 2 The proportion of β-galactosidase-positive cells (senescent cells) in the UVB-irradiated model group increased significantly compared with the untreated blank control group, indicating that the model was successfully established and can be used to detect Arabidopsis thaliana. ​ Anti-aging effect of mutant extracts; Arabidopsis thaliana ​ The proportion of β-galactosidase-positive cells (senescent cells) in the mutant extract experimental group was significantly lower than that in the model group, decreasing by 76.47% ( p<0.001 The proportion of β-galactosidase-positive cells (senescent cells) in the experimental group with wild-type Arabidopsis extract was significantly reduced compared with the model group, decreasing by 61.09% ( p<0.001 ). Add Arabidopsis ​ Compared with the experimental group 1 with wild-type Arabidopsis extract, the experimental group 2 with mutant extract showed a more significant anti-cell aging phenotype. ​ The mutant extract has the effect of significantly inhibiting cell senescence after cells are damaged by UVB.

[0082] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing an extract of a lazy mutant of Arabidopsis thaliana, characterized in that: The preparation method comprises the following steps: (1) Collecting intact plants of the Arabidopsis thaliana lazy mutant that have not bolted, washing and drying them, and obtaining dried Arabidopsis thaliana lazy mutants; wherein: The drying method is: first drying at 100-105°C for 15-20 minutes, and then drying at 60-65°C for 4-5 hours; The lazy mutant is a mutant that causes the lazy gene to be unable to express normally; (2) crushing the dried Arabidopsis lazy mutant, mixing it with a solvent, and then performing ultrasonic extraction and filtering in sequence to obtain an extract; wherein; The solvent is ethanol; (3) The extract is rotary evaporated to remove the solvent, thereby obtaining the Arabidopsis lazy mutant extract.

2. The method for preparing the Arabidopsis thaliana lazy mutant extract according to claim 1, characterized in that: In step (1), the number of cleanings is 2 to 3 times.

3. The method for preparing the Arabidopsis thaliana lazy mutant extract according to claim 1, characterized in that: In step (2), the average particle size of the crushed product is 200-250 mesh.

4. The method for preparing the Arabidopsis thaliana lazy mutant extract according to claim 1, characterized in that: In step (2): The mass ratio of the dried Arabidopsis lazy mutant to the solvent is 1:10-12; And / or, the ultrasonic extraction method is: extraction at 25-45° C. and an ultrasonic power of 2000-2200 W for 0.5-1 h.

5. The method for preparing the Arabidopsis thaliana lazy mutant extract according to claim 1, characterized in that: In step (3), the rotary evaporation method is: first, rotary evaporation at 35-42°C and 80-120 r / min until the liquid does not overflow, then raising the rotary evaporation temperature to 48-52°C, and rotary evaporation for 30-40 minutes until the solvent is completely removed.

6. An Arabidopsis thaliana lazy mutant extract obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the Arabidopsis thaliana lazy mutant extract according to claim 6 in the preparation of anti-skin aging products.

8. The use according to claim 7, characterized in that The anti-skin aging product is a product that protects telomeres; And / or, the anti-skin aging product is a product that enhances mitochondrial activity; and / or, the anti-skin aging product is a product that increases the collagen content of ECM fibers; And / or, the anti-skin aging product is an anti-cell aging product.

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