Preparation and application of cell-grade anti-aging millettia red camellia callus filtrate

Abstract

The invention discloses a preparation method and an application of a cell-grade anti-aging millettia red camellia callus filtrate. Belongs to the technical field of cosmetic materials and application thereof. The method comprises the following steps: selecting buds or flowers of camellia japonica as explants; carrying out callus induction culture on the sterilized explant; selecting the callus which is obtained by induction and is loose in texture to carry out multiplication culture; and filtering to obtain a red camellia callus cell culture filtrate. According to the method disclosed by the invention, a solid culture medium and a liquid culture medium are simultaneously used for culturing the calluses of the red camellia, and a liquid culture medium formula is preferably selected for regulating and controlling active ingredients (rutin and the like) of the calluses, so that the red camellia callus filtrate which is rich in active ingredients and can realize high addition is finally obtained; the cell-level anti-wrinkle and repairing effects are realized.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic materials and their application technology, specifically relating to the preparation and application of a filtrate of Camellia chrysantha callus with cellular-level anti-aging properties. Background Technology

[0002] Camellia chekiangoleosa Hu., a species of evergreen shrub or small tree belonging to the genus Camellia in the family Theaceae, is prized for its vibrant flowers and rarity. Ancient Camellia plants, especially those over a thousand years old, have undergone long-term natural selection and contain a variety of active substances, including flavonoids and polyphenols. Modern research indicates that these active ingredients possess skincare benefits such as antioxidant, anti-inflammatory, soothing, skin barrier repair promotion, and collagen loss delay, making them highly valuable in the cosmetics raw material field. Traditional extraction methods rely on the flowers, leaves, and seeds of natural Camellia chekiangoleosa. However, ancient Camellia chekiangoleosa plants are scarce and have a long growth cycle (flowering for only 1-2 months per year). Therefore, developing an extraction technology that does not rely on natural plants, is not limited by the growth cycle, and can efficiently enrich the active ingredients of ancient Camellia chekiangoleosa flowers is of great significance for protecting rare resources and expanding the sources of high-end skincare raw materials.

[0003] CN 112842955 A discloses the use of camellia (Camellia japonica) callus extract in the preparation of a skin care composition. The method involves creating wounds on the surface of the leaves and stems of the camellia plant by incision, tearing, or cutting to induce the formation of white granular callus. A fresh induction medium is then used to culture the callus at 25°C and 60-80% humidity. The camellia callus is freeze-dried, pulverized, and mixed with water (or C1-C4 alcohols), extracted, and filtered to remove the callus powder, yielding a filtrate. After cooling, the camellia callus extract is obtained. This skin care composition is used to reduce transepidermal water loss, reduce skin melanin content, reduce skin sagging, reduce skin texture, reduce wrinkles, and / or reduce skin pores. However, CN 112842955 A collected camellia callus tissue and freeze-dried it, then used a solvent to extract the camellia callus tissue juice, without paying attention to the callus cell culture in a culture medium containing more active ingredients. Summary of the Invention

[0004] In view of the problems described above, this invention provides a method for preparing and applying a filtrate of ancient camellia flower callus with cellular-level anti-aging properties. The number of camellia trees (in Zhejiang) with an age of over a thousand years is extremely limited, making it impractical to collect their flowers for extracts in cosmetics. This invention utilizes callus technology to cultivate flowers from ancient (Zhejiang) camellia trees. In addition to using a solid culture medium, an optimized liquid culture medium is used to regulate and induce the active ingredient rutin in the callus, ultimately yielding a filtrate of ancient camellia flower callus with a high rutin content and cellular-level anti-wrinkle effects, which can also be added to cosmetics.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] <First Aspect> This invention relates to a method for preparing a filtrate from red camellia callus tissue, the method comprising the following steps: S1. Select the flower buds or flowers of red camellia as explants; S2. Sterilize the explants and induce callus culture. S3. Select the loosely textured callus tissue obtained in step S2 for proliferation culture; S4. Filter and collect the filtrate to obtain the filtrate of red camellia callus cell culture.

[0007] As one implementation scheme, in step S2, the generation frequency is 20-30 days.

[0008] In one implementation, in step S3, the proliferation culture is a liquid medium with the following composition: MS + 0.5-1.0 mg / L TDZ + 0.5-1.0 mg / L NAA + 0.5-1.0 mg / L 6-BA + 0-0.05 M methyl jasmonate + 0-1.0 g / L phenylalanine + 0-1.0 g / L tyrosine + 0-0.5 g / L vitamin C + 20-40 g / L sucrose. In some implementation examples, the liquid medium composition is: MS + 0.8 mg / L TDZ + 0.5 mg / L NAA + 0.7 mg / L 6-BA + 0.03 M methyl jasmonate + 1.0 g / L phenylalanine + 0.5 g / L tyrosine + 0.3 g / L vitamin C + 30 g / L sucrose.

[0009] As one implementation, step S4 further includes the step of preparing one or more of p-hydroxyacetophenone, 1,2-hexanediol, 1,2-pentanediol, and octyl glycol in the camellia callus cell culture filtrate to obtain the camellia callus filtrate.

[0010] As one implementation, the total content of one or more of p-hydroxyacetophenone, 1,2-hexanediol, 1,2-pentanediol, and octyl glycol in the camellia callus filtrate is 0.1%-2.0%.

[0011] As one implementation scheme, the red camellia is a Zhejiang red camellia with a tree age of over a thousand years.

[0012] As one implementation scheme, the induction medium in step S2 can be a conventional solid culture medium. For example, the composition of the solid culture medium could be: MS + 0.5-1.0 mg / L TDZ + 0.5-1.0 mg / L NAA + 0.5-1.0 mg / L 6-BA + 20-40 g / L sucrose + 5-10 g / L agar, pH 5.84-5.87. In some implementation examples, the selected induction solid culture medium composition is: MS + 1 mg / L TDZ + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar, pH 5.84-5.87. The culture conditions are a dark, light-protected environment at a temperature of 25 ± 2°C and humidity of 60 ± 10%, equipped with air conditioning and an air purification system, ensuring the culture room has clean, sterile, and naturally circulating air.

[0013] As one implementation scheme, in step S3, the propagation culture conditions are: rotation speed 100-150 rpm, temperature 25-30 ℃, light intensity 1500-2500 lx; ​​subculture or harvesting frequency is 8-12 days.

[0014] <Second aspect> The present invention also relates to a camellia callus filtrate prepared by the aforementioned method.

[0015] As one embodiment, the filtrate contains 40-80 ppm rutin, 40-80 μg / mL total flavonoids, and 30-80 μg / mL total polyphenols. In one example, the filtrate from red camellia callus contains 65.98 ppm rutin, 64.21 μg / mL total flavonoids, and 41.16 μg / mL total polyphenols.

[0016] <Third aspect> This invention also relates to the application of red camellia callus filtrate as an anti-wrinkle ingredient in cosmetics.

[0017] The camellia callus filtrate of the present invention can be added in high quantities to cosmetics. As one embodiment, the amount of camellia callus filtrate added to the cosmetic is ≤100%.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Solid + liquid culture medium was used to culture the callus tissue of Camellia japonica at the same time, and a liquid culture medium formula under certain conditions was selected to regulate the active ingredients (rutin, etc.) of the callus tissue. Finally, a Camellia japonica callus tissue filtrate rich in active ingredients was obtained, which has the effects of cell-level anti-wrinkle and repair. (2) Select red camellias with a thousand-year-old tree and culture their callus tissue. This solves the problem of biological scarcity and enables efficient, large-scale and seasonal asexual reproduction, resulting in stable and safe cosmetic raw materials. Detailed Implementation

[0019] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0020] Examples 1-3 Examples 1-3 provide a method for preparing and applying a filtrate of *Camellia sinensis* callus with cellular-level anti-aging properties; mainly including: 1. Induction of explants from the thousand-year-old red camellia; Fresh, healthy *Camellia sinensis* flowers were selected as explants for the experiment. They were first soaked in a dish soap solution (dish soap:water = 1:200) for 10 minutes, then soaked in a 2 g / L carbendazim solution for 30 minutes. After soaking, the explants were rinsed with running water for 30 minutes to thoroughly remove the carbendazim. In a clean bench, an alcohol lamp was lit, and the explants were disinfected with 75% alcohol for 50-60 seconds. They were then rinsed three times with sterile ultrapure water, soaked in a 2% sodium hypochlorite solution (Tween 20:2% sodium hypochlorite solution = 1:500, with a small amount of Tween 20 added), and disinfected by occasional shaking for 7-10 minutes. Finally, they were rinsed 3-5 times with sterile ultrapure water to complete the surface disinfection of the explants. After blotting the surface moisture of the explants with sterile filter paper, remove any tissue that has come into contact with the disinfectant. Cut the explants into small pieces and inoculate them onto solid culture medium (MS + 1 mg / L TDZ + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar, pH 5.84-5.87) for callus induction. Change the culture medium regularly according to the callus growth, with a preferred subculture (medium change) frequency of 25 days.

[0021] 2. Preparation of culture medium; Single-factor experiments were used to screen the inducing factors required in the culture medium formulation for plant callus tissue, obtaining the optimal process conditions for the liquid culture medium. The liquid culture medium formulation was optimized, and the pH was adjusted to 5.84-5.87. Liquid culture of Camellia chrysantha callus tissue was then performed under these conditions. The liquid culture medium formulations for each example and comparative example are as follows: Control example (initial): MS + 1 mg / L TDZ + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 30 g / L sucrose; Example 1: MS + 0.5 mg / L TDZ + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 0.02 M methyl jasmonate + 0.5 g / L tyrosine + 30 g / L sucrose; Example 2: MS + 0.5 mg / L TDZ + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 0.03 M methyl jasmonate + 1.0 g / L phenylalanine + 0.3 g / L vitamin C + 30 g / L sucrose; Example 3: MS + 0.8 mg / L TDZ + 0.5 mg / L NAA + 0.7 mg / L 6-BA + 0.03 M methyl jasmonate + 1.0 g / L phenylalanine + 0.5 g / L tyrosine + 0.3 g / L vitamin C + 30 g / L sucrose.

[0022] 3. Preparation of filtrate from the callus cell culture of Camellia chrysantha; Liquid culture media were prepared according to the above-mentioned culture medium formula and dispensed into 250 mL Erlenmeyer flasks, 100 mL per flask. Loose callus tissue was selected, picked up with forceps, and evenly dispersed in the liquid culture medium. 10 g of callus tissue was added to each flask. The flasks were placed in a horizontal shaker and cultured under the following conditions: 130 rpm, 25 ℃, and 1500 lx light intensity. The preferred subculture or harvesting frequency was 10 days to obtain the *Camellia chrysantha* callus cell culture. The culture was filtered to obtain the *Camellia chrysantha* callus cell culture filtrate.

[0023] 4. Preparation of callus filtrate from *Camellia chrysantha* var. *mairei*; The filtrate of the callus cell culture of Camellia chrysantha was obtained, and 0.4% p-hydroxyacetophenone, 0.5% 1,2-hexanediol and 0.5% 1,2-pentanediol were used to prepare the Camellia chrysantha callus filtrate.

[0024] Test Example 1: Analysis of Active Components in the Filtrate of Camellia japonica Callus Cell Culture The contents of rutin, total flavonoids, and total polyphenols in the filtrate of the callus cell culture of Camellia chrysantha were determined, and the results are shown in Table 1.

[0025] 1. Rutin content detection; Test Principle: High-performance liquid chromatography (HPLC) is based on the physicochemical properties of active substances, converting these properties into a detector response signal, which is then characterized by a chromatogram. Rutin has a characteristic peak at 360 nm, which can be used for rutin content detection. Test method: Weigh an appropriate amount of rutin and dissolve it in methanol to prepare a standard solution with a concentration of 1 mg / mL. Pipette 1 mL of the standard solution using a 1 mL syringe and filter it through a 0.22 µm syringe filter for HPLC detection. Pipette 1 mL of the *Camellia sinensis* callus filtrate using a 1 mL syringe and filter it through a 0.22 µm syringe filter. Pour the filtrate into a brown liquid chromatography vial for HPLC detection.

[0026] 2. Detection of total flavonoid content; Test principle: The benzopyran ring of flavonoids undergoes a complexation reaction with aluminum salts to form a yellow complex under alkaline conditions. The absorbance of the solution is measured at an absorption wavelength of 500 nm, and the total flavonoid content is quantified using rutin as a calibration standard. Test method: Take 3 mL of sample solution, add 0.5 mL of 5% sodium nitrite and 0.5 mL of 10% aluminum nitrate in sequence (mix well after each step and let stand at room temperature for 6 min), then add 5 mL of 4% sodium hydroxide (mix well and let stand at room temperature for 15 min). Zero the instrument with a blank tube (add 3 mL of distilled water) and measure the absorbance at 500 nm. Finally, test a series of standard solutions with different rutin contents. Plot a standard curve with rutin content as the abscissa and absorbance as the ordinate. Substitute the absorbance of the test sample into the curve equation to calculate the total flavonoid content (unit: μg / mL).

[0027] 3. Detection of total polyphenol content; Test principle: Folin-Ciocalteu reagent oxidizes the -OH groups in polyphenols and turns blue. The absorbance of the solution is measured at an absorption wavelength of 765 nm. Gallic acid is used as a calibration standard to quantify the total polyphenol content. Test method: Prepare a series of standard curve solutions with different gallic acid contents; for testing, take 1 mL of standard curve solution (or sample solution), add 5 mL of 10% Folin-Ciocalteu reagent, shake to mix, and after reacting for 5 min, add 4 mL of 7.5% N2 solution within 3 min. a2 CO3 was used to react the sample in the dark for 1 hour. The absorbance of the reaction solution was then measured at 765 nm in a 96-well plate.

[0028] Table 1

[0029] Test Example 2: Safety Evaluation of Camellia japonica callus filtrate 1. Chicken embryo chorioallantoic membrane experiment; This is an earlier-used in vitro method for assessing eye irritation. The chorioallantoic membrane (CAM) is a respiratory membrane surrounding the chicken embryo. This experiment utilizes the intact, clear, and transparent vascular system of the mid-stage chorioallantoic membrane in hatched chicken embryos. A certain amount of the test substance is directly contacted with the chorioallantoic membrane, and after a period of time, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and vascularization) are observed. These indicators reflect changes in the morphology, color, and permeability of blood vessels and vascular networks, as well as phenomena such as chorioallantoic membrane protein denaturation and the degree of damage. These indicators are then combined to obtain a score used to assess the eye irritation of the *Camellia sinensis* callus filtrate.

[0030] According to the ES values, the eye irritation of the test substances was classified as shown in Table 2. Table 3 shows that 100% of the *Camellia sinensis* callus filtrate was non-irritating to the eyes.

[0031] Table 2. Evaluation of Endpoint Scoring Method Results

[0032] Table 3. Stimulus scores (end point score, ES) of the test substance

[0033] 2. Human skin patch test; Using qualified patch testing equipment, an occlusive patch test was conducted. Approximately 0.020 mL to 0.025 mL of the test substance was placed in the patch device, and a hypoallergenic adhesive tape was applied to the inside of the subject's arm. The test substance was removed after 24 hours, and skin reactions were observed at 0.5, 24, and 48 hours after removal. The results were recorded according to the skin reaction grading standards in the "Cosmetic Safety Technical Specifications" (2015 edition). The results are shown in Table 4. No adverse reactions were observed in any of the 30 participants tested; therefore, the product passed the human skin patch test.

[0034] Table 4 Patch Test Results

[0035] Test Example 3: Evaluation of the Cellular-Level Anti-Aging Efficacy of Camellia japonica Callus Fragrance 1. Cytotoxicity of Camellia chrysantha callus filtrate on different cell types; Cell viability is commonly used as an indicator of a component's cytotoxicity, while the CCK-8 assay can determine the number of viable cells, offering advantages such as high sensitivity and no radioactivity. The results are shown in Table 5. Test method: A sample group and a blank control group were set up for comparative experiments. The cell experiments were performed in 6 replicates (n=6). Logarithmic growth phase cells were collected and processed at a ratio of 1×10⁻⁶. 4 Cells were seeded per well in a 96-well plate and incubated at 37 °C with 5% CO2 for 24 h. The supernatant was discarded, and the cells were washed twice with PBS. The samples were dissolved in DMEM complete culture medium to prepare solutions with corresponding concentration gradients, 100 pL per well, and incubated for 24 h. After adding 10 µL of LCK-8 reagent and culturing for 1 h, the OD value at 450 nm was measured. The cell viability calculation formula is shown below: ; Where: V(%)—cell viability, %; OD sample—absorbance of the reaction system containing the test sample; OD empty plate—absorbance of the empty plate without any substance; OD blank control—OD empty plate; OD blank control—absorbance of the reaction system without the test sample.

[0036] Table 5

[0037] 2. Effects of Camellia chrysantha callus filtrate on the expression of anti-wrinkle marker genes; Principle: The COLIA1 gene encodes the α1 chain of type I collagen, a major component of type I collagen. Type I collagen is the most abundant type of collagen in the human body, widely found in connective tissues such as skin, bones, tendons, and ligaments. Type I collagen is a major component of the dermis, forming collagen fibers and providing elasticity and firmness to the skin. It plays a crucial role in maintaining the structural integrity and strength of the skin. With age, the expression of the COLIA1 gene and the rate of type I collagen synthesis decrease, while its degradation rate increases. This change leads to the breakage and reduction of collagen fibers, resulting in signs of aging such as sagging skin, wrinkles, and dryness.

[0038] The SIRT3 gene encodes an NAD+-dependent deacetylase, primarily located in the mitochondrial matrix, and is widely recognized as a "mitochondrial longevity protein." It regulates various metabolic enzymes and antioxidant proteins through deacetylation, and also promotes mitochondrial biogenesis (PGC-1α pathway) and mitophagy, clears damaged mtDNA, and maintains mitochondrial network homeostasis.

[0039] The SIRT7 gene encodes an NAD*-dependent nucleolar deacetylase, primarily located in the nucleolus and chromatin. Studies have shown that SIRT7 can influence p53 activity by maintaining genome stability, thereby regulating cellular senescence. SIRT7 overexpression reduces the expression levels of p53, p21, and γH2AX, an early marker of DNA damage, thus delaying premature cellular senescence.

[0040] Specific experimental steps: (1) HDF cells in the logarithmic growth phase were fed with 1*10 6 Cells were seeded per well in 6-well plates and incubated at 37°C with 5% CO2 for 24 hours. (2) After UVA irradiation of 15 J / cm², samples were added to the sample group, and culture medium containing 10% serum was added to the blank control group and the model group. The samples were incubated at 37℃ and 5% CO2 for 24 h (the blank control group did not need UVA irradiation). (3) Discard the supernatant, wash twice with PBS, and then collect the cells; (4) RNA was extracted, reverse transcribed into cDNA, and then subjected to RT-qPCR; (5) The relative expression level of the target gene was determined using GAPDH as an internal reference, through 2 -△△Ct The method is used for calculation; 2 -△△Ct =2 -[(实验组目的基因CT-实验组内参基因CT)-(对照组目的基因CT-对照组内参基因CT)] ; (6) Import the calculated values ​​into GraphpadPrism8, perform statistical analysis and plot the graph.

[0041] The results are shown in Tables 6, 7, and 8. Compared with the control group, after UVA irradiation, the expression levels of COLIA1, SIRT3, and SIRT7 decreased by 55.75% (P<0.001), 38.39% (P<0.01), and 44.60% (P<0.001), respectively, indicating the effectiveness of the model. Compared with the model group, 2% Camellia japonica callus filtrate significantly promoted the expression of COLIA1, SIRT3, and SIRT7 genes, with increases of 33.31%, 48.19%, and 30.20%, respectively. These results indicate that 2% Camellia japonica callus filtrate has a cellular-level anti-wrinkle and firming effect.

[0042] Table 6 Analysis of COLIAI gene expression results

[0043] Table 7 Analysis of SIRT3 gene expression results

[0044] Table 8. Analysis of SIRT7 gene expression results

[0045] 3. Effects of Camellia chrysantha callus filtrate on the expression of repair marker genes; The TGMI gene encodes transglutaminase 1, a calcium-dependent membrane-bound enzyme. This enzyme cross-links various structural proteins to form a keratinized capsule and covalently links long-chain hydroxyceramides to capsule proteins to form a lipid capsule, thereby enhancing the integrity and water resistance of the skin barrier. The LORICRIN gene encodes loricrin, a major component of the keratinized capsule that plays a crucial role in the barrier function of the epidermis. Catalyzed by transglutaminases (such as TGM1), loricrin cross-links with other capsule proteins to form a stable, water-insoluble structure, thus constructing a tough keratinized capsule that provides the stratum corneum with mechanical strength and barrier function. The HAS2 gene encodes hyaluronic acid synthase 2, responsible for synthesizing hyaluronic acid, a polysaccharide that plays multiple functions in the extracellular matrix, including space filling, joint lubrication, and providing a matrix for cell migration. Hyaluronic acid is actively produced during wound healing and tissue repair, providing a framework for the intracellular growth of blood vessels and fibroblasts. Therefore, this experimental project selected the expression of TGMI, LORICRIN, and HAS2 genes as key indicators for evaluating repair efficacy.

[0046] Specific experimental steps: (1) HaCaT cells in the logarithmic growth phase were fed with 1*10 6 Cells were seeded per well in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. (2) After UVB irradiation at 60 mJ / cm², samples were added to the sample group, while culture medium containing 10% serum was added to the blank control group and the model group. The samples were then incubated at 37°C. Incubate in a 5% CO2 incubator for 24 hours (the blank control group does not require UVB irradiation); (3) Discard the supernatant, wash twice with PBS, and then collect the cells; (4) RNA was extracted, reverse transcribed into cDNA, and then subjected to RT-qPCR; (5) The relative expression level of the target gene was determined using GAPDH as an internal reference, through 2 -△△Ct The method is used for calculation; (6) Import the calculated values ​​into GraphpadPrism8, perform statistical analysis and plot the graph.

[0047] As shown in Tables 9, 10, and 11, compared with the control group, UVB irradiation significantly reduced the expression levels of TGM1, LORICRIN, and HAS2 genes by 36.67% (P<0.01), 28.69% (P<0.05), and 35.92% (P<0.01), respectively, indicating the model's effectiveness. Compared with the model group, 100 ppm of asiaticoside significantly increased the expression levels of TGM1, LORICRIN, and HAS2 genes by 100.99% (P<0.001), 44.62% (P<0.05), and 51.42% (P<0.01), respectively, demonstrating the effectiveness of the positive reference. Compared with the model group, 2% Camellia chrysantha callus filtrate increased the expression levels of TGM1, LORICRIN and HAS2 genes by 252.92% (P<0.001), 148.72% (P<0.001) and 112.55% (P<0.001), respectively, indicating that 2% Camellia chrysantha callus filtrate has a cell-level repair effect.

[0048] Table 9. Analysis of TGM1 gene expression results

[0049] Table 10 Analysis of LORICRIN gene expression results

[0050] Table 11 Analysis of HAS2 gene expression results

[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a filtrate from red camellia callus tissue, characterized in that, The method includes the following steps: S1. Select the flower buds or flowers of red camellia as explants; S2. Sterilize the explants and induce callus culture. S3. Select the loosely textured callus tissue obtained in step S2 for proliferation culture; S4. Filter and collect the filtrate to obtain the filtrate of red camellia callus cell culture.

2. The method for preparing camellia callus filtrate according to claim 1, characterized in that, In step S2, the succession frequency is 20-30 days.

3. The method for preparing camellia callus filtrate according to claim 1, characterized in that, In step S3, the proliferation culture is a liquid culture medium with the following composition: MS + 0.5-1.0 mg / L TDZ + 0.5-1.0 mg / L NAA + 0.5-1.0 mg / L 6-BA + 0-0.05 M methyl jasmonate + 0-1.0 g / L phenylalanine + 0-1.0 g / L tyrosine + 0-0.5 g / L vitamin C + 20-40 g / L sucrose.

4. The method for preparing camellia callus filtrate according to claim 1, characterized in that, Step S4 is followed by a step of preparing one or more of p-hydroxyacetophenone, 1,2-hexanediol, 1,2-pentanediol, and octyl glycol in the filtrate of red camellia callus cell culture to obtain red camellia callus filtrate.

5. The method for preparing camellia callus filtrate according to claim 4, characterized in that, The total content of one or more of p-hydroxyacetophenone, 1,2-hexanediol, 1,2-pentanediol, and octyl glycol in the camellia callus filtrate is 0.1%-2.0%.

6. The method for preparing camellia callus filtrate according to claim 1, characterized in that, The red camellia mentioned is a Zhejiang red camellia with a tree age of over a thousand years.

7. A camellia callus filtrate prepared by the method according to any one of claims 1-6.

8. The camellia callus filtrate according to claim 7, characterized in that, The filtrate contains 40-80 ppm rutin, 40-80 μg / mL total flavonoids, and 30-80 μg / mL total polyphenols.

9. The use of the camellia callus filtrate as described in claim 7 as an anti-wrinkle ingredient in cosmetics.

10. The application according to claim 9, characterized in that, In the cosmetic product, the amount of red camellia callus filtrate added is ≤100%.

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