Multi-effect application of bletilla chinensis callus extract in field of cosmetics

Abstract

The invention provides multi-effect application of bletilla chinensis callus extract in the field of cosmetics, and belongs to the technical field of cosmetic raw material development. The invention provides application of bletilla chinensis callus extract in resisting aging, repairing skin barrier, moisturizing, relieving, resisting light aging, repairing DNA and balancing cell cycle, the extract has rare bibenzyl substances, such as 3, 3 '-dihydroxy-2-p-hydroxybenzyl-5-methoxy bibenzyl, 3, 3', 5-trihydroxy bibenzyl, 3, 3 '-dihydroxy-5-methoxy bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl, dimethoxyl bibenzyl The compound is prepared from the following raw materials: 3, 3 '-dihydroxy-5-methoxy bibenzyl), dendrophenol (3, 3'-dihydroxy-5, 4 '-dimethoxy bibenzyl), Dactylorin E and the like; the bletilla chinensis callus extract has excellent effects in the aspects of aging resistance, skin barrier repairing, soothing, light aging resistance, DNA repairing and cell cycle balancing, the application of the bletilla chinensis callus extract in cosmetics is developed, a new raw material is provided for the field of cosmetics, and the bletilla chinensis callus extract has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material development technology, and in particular to the multifunctional applications of Hua Bai and callus extracts in the cosmetic field. Background Technology

[0002] Plant-based ingredients refer to active components extracted from plants, processed using modern techniques, and then added to skincare products for use in the preparation of cosmetics with specific effects. Compared to traditional chemical-based skincare products, those with plant extracts as the main ingredient have advantages such as being environmentally friendly, having traceable and safe sources, and offering a wide range of functions, making them a current research hotspot in the cosmetics field.

[0003] Bletilla sinensis (Rolfe) Schltr., commonly known as Chinese Bletilla, is a perennial herbaceous plant belonging to the Orchidaceae family. It typically grows on rock walls or in crevices. Due to its low natural fruit set and demanding growth and reproduction conditions, it had not been found in the wild for over 120 years until its rediscovery in Yunnan Province in 2021. Chinese Bletilla is currently listed as an endangered species on the IUCN Red List and is listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

[0004] Due to the scarcity of Chinese white herb germplasm resources, its scientific research and production applications are limited. There is currently no technological research or effective application in the cosmetics field. How to transform it into high-efficacy cosmetic products remains to be further explored. Summary of the Invention

[0005] The purpose of this invention is to provide multifunctional applications of Hua Bai and callus extracts in the cosmetics field, providing excellent raw materials for the cosmetics industry.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of Huabai and callus extracts in the preparation of anti-aging products.

[0007] This invention also provides the application of Hua Bai and callus extracts in the preparation of products that repair the skin barrier.

[0008] This invention also provides the application of Hua Bai and callus extracts in the preparation of moisturizing products.

[0009] This invention also provides the application of Hua Bai and callus extracts in the preparation of soothing products.

[0010] This invention also provides the application of Huabai and callus extracts in the preparation of anti-photoaging products.

[0011] This invention also provides the application of Huabai and callus extracts in the preparation of DNA repair products.

[0012] This invention also provides the application of Huabai and callus extracts in the preparation of balanced cell cycle products.

[0013] Preferably, the product is a cosmetic.

[0014] Preferably, the preparation method of the Huabai and callus extract includes the following steps: The callus tissue and the white chrysanthemum were mixed with an alcohol reagent and subjected to alcohol extraction to obtain the alcohol extract. The alcohol extract was subjected to rotary evaporation to obtain the rotary evaporation product; The rotary evaporation product was subjected to enzymatic hydrolysis to obtain the enzymatic hydrolysis product, which is the extract of Hua Bai and callus tissue. The alcohol reagent is ethanol, and the volume percentage concentration of the ethanol is 50-99%. The weight ratio of the white chrysanthemum and callus tissue to the volume ratio of the alcohol reagent is 1g:3~8L; The alcohol extraction process is accompanied by an ultrasonic environment, the power of which is 2000~3000W. The alcohol extraction process is repeated 3 to 8 times, with each extraction lasting 20 to 100 minutes and an interval of 20 to 40 minutes. The temperature for the alcohol extraction treatment is 20~40℃; The alcohol extract is further filtered sequentially through a Buchner funnel and a 0.22 µm filter membrane before being subjected to rotary evaporation. The rotary evaporation process includes first processing at 30~40℃ and 50~150rpm, and then processing at 40~60℃ and 100~200rpm. The enzyme reagent used in the enzymatic hydrolysis treatment is protease; The enzymatic hydrolysis process includes the following steps: The rotary evaporation product was mixed with water and protease, incubated, and then subjected to enzyme inactivation treatment. The volume ratio of the rotary evaporation product to water to the weight of the protease is 1~3:20~40g; The incubation temperature is 40~50℃, and the time is 1~3h; The enzyme inactivation treatment is carried out at 80-90°C for 10-20 minutes. And / or, the enzymatic hydrolysis product further includes filtration through a 0.22 µm filter membrane; The enzymatic hydrolysis products also undergo freeze-drying treatment; Before the Huabai and callus tissue are mixed with the alcohol reagent, they undergo a mixed liquid nitrogen grinding process. The Huabai and callus tissue are Huabai and callus tissue dry powder; The target particle size for the mixed liquid nitrogen grinding treatment is ≤40 mesh.

[0015] Preferably, the preparation method of the Huabai and callus extract includes the following steps: The callus tissue, ethyl acetate, and β-cyclodextrin were mixed and extracted with ethyl acetate to obtain the ethyl acetate extract. The ethyl acetate extract was subjected to rotary evaporation to obtain the rotary evaporation product, which is the extract of Huabai and callus tissue. The weight ratio of the white chrysanthemum and callus tissue to the volume ratio of ethyl acetate is 1g:8~12L; The weight ratio of the white fungus and callus tissue to β-cyclodextrin is 10~30:1; The ethyl acetate extraction process is performed under an ultrasonic environment with a power of 1500~3000W. The ethyl acetate extraction process is performed 3 to 8 times, with each extraction lasting 20 to 100 minutes and an interval of 20 to 40 minutes. The temperature for the ethyl acetate extraction treatment is 20~40℃; The ethyl acetate extract is further filtered sequentially through a Buchner funnel and a 0.22 µm filter membrane before being subjected to rotary evaporation. The rotary evaporation process includes first processing at 30~40℃ and 80~110rpm, then processing at 40~45℃ and 110~130rpm, and finally processing at 45~50℃ and 130~180rpm. Before the Huabai and callus tissue are mixed with the alcohol reagent, they undergo a mixed liquid nitrogen grinding process. The Huabai and callus tissue are Huabai and callus tissue dry powder; The target particle size for the mixed liquid nitrogen grinding treatment is ≤40 mesh.

[0016] The beneficial effects of this invention are: This invention provides the application of *Hypericum perforatum* and callus extract in anti-aging, skin barrier repair, moisturizing, soothing, anti-photoaging, DNA repair, and cell cycle balancing. The extract contains rare bibenzyl substances such as 3,3'-dihydroxy-2-p-hydroxybenzyl-5-methoxybibenzyl, 3,3',5-trihydroxybibenzyl, 3,3'-dihydroxy-5-methoxybibenzyl, yam extract III (3,3'-dihydroxy-5-methoxybibenzyl), dendrobine (3,3'-dihydroxy-5,4'-dimethoxybibenzyl), and Dactylorhin E. It achieves excellent efficacy in anti-aging, skin barrier repair, soothing, anti-photoaging, DNA repair, and cell cycle balancing. This invention expands the application of *Hypericum perforatum* and callus extract in cosmetics, providing a new raw material for the cosmetics field with broad application prospects. Attached Figure Description

[0017] Figure 1 Schematic diagram of total ion flow of chemical components in extracts of Cosmos bicolor and callus tissue; Figure 2 A graph showing the percentage of chemical substances in extracts of Cosmos bicolor and callus tissue; Figure 3 Figure showing the results of upregulating the expression of apoptosis-related genes by extracts of Codonopsis pilosula and callus tissue; Figure 4 The results of upregulating the expression of DNA mismatch repair-related genes by extracts of Codonopsis pilosula and callus tissue; Figure 5 Figure showing the results of upregulating the expression of genes related to the PI3K-Akt signaling pathway by extracts of Codonopsis pilosula and callus tissue; Figure 6 Figure showing the results of upregulating the expression of genes related to the cAMP signaling pathway using extracts from Columbarium quinquefolium and callus tissue; Figure 7 The results of upregulating the expression of HGF and FGF1 growth factors by extracts of Codonopsis pilosula and callus tissue; Figure 8 Figure showing the results of upregulating the expression of genes related to collagen synthesis by extracts of Collagen Fibrosum and callus tissue; Figure 9 Figure showing the results of upregulating the expression of the SRC gene in cells by extracts of Codonopsis pilosula and callus tissue; Figure 10 Figure showing the effect of extracts from Cosmodium styracifolium and callus tissue on the expression of skin barrier-related genes; Figure 11 Figure showing the results of downregulating the expression of cellular inflammation-related genes by extracts of Columbarium affine and callus tissue; Figure 12 The results of upregulating the expression of DNA photodamage repair-related genes by extracts of Cosmos bipinnatifida and callus tissue; Figure 13Figure showing the results of upregulating the expression of cell growth cycle-related genes by extracts of Codonopsis pilosula and callus tissue; Figure 14 A statistical graph showing the difference in TEWL values ​​of skin after treatment with 1% Cosmopolitan and callus extract samples; Figure 15 A statistical chart showing the difference in skin hemoglobin values ​​after treatment with 1% Cosmophagocytosis and callus extract samples. Detailed Implementation

[0018] This invention provides the application of Huabai and callus extracts in the preparation of anti-aging products.

[0019] This invention also provides the application of Hua Bai and callus extracts in the preparation of products that repair the skin barrier.

[0020] This invention also provides the application of Hua Bai and callus extracts in the preparation of moisturizing products.

[0021] This invention also provides the application of Hua Bai and callus extracts in the preparation of soothing products.

[0022] This invention also provides the application of Huabai and callus extracts in the preparation of anti-photoaging products.

[0023] This invention also provides the application of Huabai and callus extracts in the preparation of DNA repair products.

[0024] This invention also provides the application of Huabai and callus extracts in the preparation of balanced cell cycle products.

[0025] Preferably, the product is a cosmetic.

[0026] In this invention, the anti-aging product refers to a product that works by slowing down skin aging, for example by promoting the expression of growth factor genes HGF and FGF1, promoting the expression of collagen synthesis-related genes LAMA2, COL4A5, COL18A1 and COL14A1, and promoting the expression of cell proliferation, migration and adhesion-related genes SRC. The skin barrier repair products refer to products that exert their effects by repairing the skin barrier function, such as enhancing barrier homeostasis and repair capabilities by upregulating the expression of PI3K-Akt signaling pathway genes PIK3R1 and RELN, as well as cAMP signaling pathway genes CACNA1C and CAMK4. The moisturizing products mentioned refer to those that work by reducing skin moisture loss, such as improving the skin's ability to retain moisture by significantly reducing transepidermal water loss (TEWL), for example, reducing TEWL values ​​by 17-19% in human tests; The soothing products refer to products that work by reducing inflammation and relieving skin irritation, such as by inhibiting the production of pro-inflammatory factors by downregulating the expression of inflammation-related genes MAPK13 and GADD45G. The aforementioned anti-photoaging products refer to products that work by defending against ultraviolet damage, such as by upregulating the expression of DNA photodamage repair genes such as UNG, TDG, MSH2, PCNA, EXO1, XPA, RPA1, MNAT1, POLE2, and POLE3 to enhance the efficiency of UV damage clearance. The DNA repair products mentioned refer to products that exert their effects by repairing DNA damage, such as by upregulating the expression of apoptosis and repair genes CASP10, PARP1, BCL2L1, GTF2H2B and GTF2H2C to enhance cellular genome stability. The aforementioned cell cycle balancing products refer to products that exert their effects by maintaining the balance between cell proliferation and apoptosis, such as by upregulating the expression of cell cycle genes CDK1, CDK2, CCNB1, PLK1, KNL1, SKP2, and E2F2 to coordinate cell division and repair processes.

[0027] Preferably, the preparation method of the Huabai and callus extract includes the following steps: The callus tissue and the white chrysanthemum were mixed with an alcohol reagent and subjected to alcohol extraction to obtain the alcohol extract. The alcohol extract was subjected to rotary evaporation to obtain the rotary evaporation product; The rotary evaporation product was subjected to enzymatic hydrolysis to obtain the enzymatic hydrolysis product, which is the extract of Hua Bai and callus tissue.

[0028] In this invention, preferably, the alcohol reagent is ethanol, and the volume percentage concentration of the ethanol is 50-99%, more preferably 65-75%; the weight ratio of the chrysophanol and callus tissue to the volume of the alcohol reagent is 1g:3-8L, more preferably 1g:4.5-5.5L; the alcohol extraction treatment is accompanied by an ultrasonic environment, and the power of the ultrasonic environment is 2000-3000W, more preferably 2400-2600W; the number of alcohol extraction treatments is 3-8 times, more preferably 4-6 times, the time of each alcohol extraction treatment is 20-100min, more preferably 45-55min, with an interval of 20-40min, more preferably 25-35min; the temperature of the alcohol extraction treatment is 20-40℃, more preferably 30-36℃; the alcohol extract product further includes passing through a Buchner funnel and a 0.22 Filtration is performed using a µm filter membrane; the rotary evaporation process includes initial treatment at 30-40°C and 50-150 rpm, more preferably 35-38°C and 90-110 rpm, followed by treatment at 40-60°C and 100-200 rpm, more preferably 47-49°C and 125-135 rpm; preferably, the enzyme used in the enzymatic hydrolysis is a protease; the enzymatic hydrolysis includes the following steps: mixing the rotary evaporation product with water and protease, incubating, and then performing enzyme inactivation treatment; The weight ratio of the rotary evaporation product to water and the protease is 1-3:20-40g, more preferably 1.4-1.6L:28-32g; the incubation temperature is 40-50℃, more preferably 44-46℃, and the time is 1-3h, more preferably 1.8-2.2h; the enzyme inactivation treatment temperature is 80-90℃, more preferably 84-86℃, and the time is 10-20min, more preferably 14-16min; and / or, the enzymatic hydrolysis product further includes filtration through a 0.22 µm filter membrane; the enzymatic hydrolysis product further includes freeze-drying.

[0029] This invention also provides another method for preparing extracts of Cosmodium and callus tissue, comprising the following steps: The callus tissue, ethyl acetate, and β-cyclodextrin were mixed and extracted with ethyl acetate to obtain the ethyl acetate extract. The ethyl acetate extract was subjected to rotary evaporation to obtain the rotary evaporation product, which is the extract of Huabai and callus tissue.

[0030] The weight ratio of the white fungus and callus tissue to β-cyclodextrin is 10~30:1; In this invention, the weight ratio of the white chrysanthemum and callus tissue to the volume of ethyl acetate is preferably 1g:8~12L, more preferably 1g:9~11L; the ethyl acetate extraction process is accompanied by an ultrasonic environment, the power of which is 1500~3000W, more preferably 1800~2200W; the number of ethyl acetate extraction processes is 3~8 times, more preferably 4~6 times, the time for each ethyl acetate extraction process is 20~100min, more preferably 25~35min, with an interval of 20~40min, more preferably 25~35min; the temperature of the ethyl acetate extraction process is 20~40℃, more preferably 30~35℃. Preferably, the ethyl acetate extract is further filtered sequentially through a Buchner funnel and a 0.22 µm filter membrane before rotary evaporation. The rotary evaporation process includes initial treatment at 30–40°C and 80–110 rpm, more preferably 33–37°C and 90–100 rpm, followed by treatment at 40–45°C and 110–130 rpm, more preferably 42–44°C and 115–125 rpm, and finally treatment at 45–50°C and 130–180 rpm, more preferably 47–49°C and 140–160 rpm. Preferably, the von Willebrand and callus tissue are subjected to mixed liquid nitrogen grinding before mixing with the alcohol reagent; the von Willebrand and callus tissue are von Willebrand and callus tissue dry powder; the target particle size of the mixed liquid nitrogen grinding process is ≤40 mesh.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The chrysophanol and callus tissue used in the examples were prepared using the following method: 1) Induction of callus from *Polygonum multiflorum*: Rinse *Polygonum multiflorum* leaves with running water for 10 min, soak in 75% alcohol for 5 min in a clean bench, then soak in 2% sodium hypochlorite solution for 10 min. Use sterile filter paper to absorb the surface moisture of the explants. Cut the *Polygonum multiflorum* leaves into small segments of about 1 cm with a sterile scalpel. Place them on 4.41 g / L MS medium + 30 g / L sucrose + 7.5 g / L agar + 2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) + 1 mg / L 6-BA (6-benzylaminopurine). Control the temperature at 23±2℃, humidity at 70-80%, and in the dark. Replace the medium with fresh medium every 20 days until pale yellow callus appears.

[0033] 2) Obtaining Corynebacterium wilfordii and callus: The Corynebacterium wilfordii and callus were picked and transferred to 4.41 g / L MS medium + 30 g / L sucrose + 7.5 g / L agar + 1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) + 1.5 mg / L 6-BA (6-benzylaminopurine) + 0.5 g / L phenylalanine. The Corynebacterium wilfordii and callus were harvested after 25 days of culture under controlled conditions of 23±2℃, 70-80% humidity and darkness.

[0034] Place the harvested chrysophanite and callus tissue in a -80℃ freezer for 2-4 hours or quick-freeze with liquid nitrogen for 30 minutes; spread the pre-frozen chrysophanite and callus tissue evenly in a freezing tray, close the door, and set the process parameters (see Table 1-0 below); primary drying (sublimation): remove free water, 20-24 hours; secondary drying: remove bound water, 4-8 hours, final moisture content <5%; the freeze-drying process is completed, and lyophilized chrysophanite and callus tissue powder is obtained.

[0035] Table 1-0 Process Parameter Table

[0036] Example 1 1) Weigh 500 g of dried powder of Huabai and callus tissue, add liquid nitrogen and grind until fine powder (≤40 mesh).

[0037] 2) Add 5 L (500g:5000mL) of 70% ethanol solution at a material-to-liquid ratio of 1:10 (m / v). Perform ultrasonic extraction at 2500 W, 30℃, for 50 min, with 30 min intervals, repeating the extraction 5 times. After extraction, pass the solution through a Buchner funnel and a 0.22 µm filter membrane to remove impurities, obtaining the ethanol extract.

[0038] 3) Transfer the above alcohol extract to a rotary evaporator at 35°C and 100 rpm to remove most of the ethanol. Increase the temperature to 48°C and evaporate at 130 rpm for 30 minutes to completely remove the ethanol.

[0039] 4) Add ultrapure water to the rotary evaporated solution and bring the volume to 1.5 L. Add 30 g of neutral protease and incubate at 45 °C for 2 h. After enzymatic hydrolysis, rapidly heat the solution to 85 °C and incubate for 15 min to completely inactivate the neutral protease.

[0040] 5) Quickly cool the above solution to room temperature (25°C), remove impurities by filtering through a 0.22 µm filter membrane, and concentrate to an anhydrous state by freeze-drying (refer to the freeze-drying method for fresh Hua Bai and callus tissues above) to obtain the alcohol extract of Hua Bai and callus tissues.

[0041] Example 2 1) Weigh 500 g of dried powder of Huabai and callus tissue, add liquid nitrogen and grind until fine powder (≤40 mesh).

[0042] 2) Add 5 L of 70% ethanol solution at a material-to-liquid ratio of 1:10 (m / v). Perform ultrasonic extraction at 2500 W, 35℃, for 60 min, with 30 min intervals, repeating the extraction 5 times. After extraction, pass the solution through a Buchner funnel and a 0.22 µm filter membrane to remove impurities, obtaining the ethanol extract.

[0043] 3) Transfer the above alcohol extract to a rotary evaporator at 40℃ and 120 rpm to remove most of the ethanol. Increase the temperature to 48℃ and 140 rpm, and evaporate for 30 min to completely remove the ethanol.

[0044] 4) Add ultrapure water to the rotary evaporated solution and bring the volume to 1.5 L. Add 30 g of neutral protease and incubate at 45 °C for 2 h. After enzymatic hydrolysis, rapidly heat the solution to 85 °C and incubate for 15 min to completely inactivate the neutral protease.

[0045] 5) The above solution was rapidly cooled to room temperature (25°C), impurities were removed by passing it through a 0.22 µm filter membrane, and the solution was concentrated to anhydrous state by freeze drying to obtain the alcohol extract of Hua Bai and callus tissue.

[0046] Example 3 1) Weigh 500 g of dried powder of Huabai and callus tissue, add liquid nitrogen and grind until fine powder (≤40 mesh).

[0047] 2) Add 5 L of ethyl acetate at a material-to-liquid ratio of 1:10 (m / v), along with 25 g of β-cyclodextrin. Perform ultrasonic extraction at 2000 W, 32℃, and 30 min for 30 min intervals, repeating the extraction four times. After extraction, pass the solution through a Buchner funnel and a 0.22 µm filter membrane to remove impurities, obtaining the ethyl acetate extract.

[0048] 3) Transfer the above ethyl acetate extract into a rotary evaporator at 35°C and 90 rpm. Then, increase the temperature to 42°C and the rotation speed to 100 rpm for 30 min to remove most of the ethyl acetate.

[0049] 4) Continue to raise the temperature to 48℃ and rotate the spindle at 130 rpm until the ethyl acetate is completely removed. After rotary evaporation, cool to room temperature to obtain the ethyl acetate extract of chrysanthemum and callus.

[0050] Example 4 1) Weigh 500 g of dried powder of Huabai and callus tissue, add liquid nitrogen and grind until fine powder (≤40 mesh).

[0051] 2) Add 5 L of ethyl acetate at a material-to-liquid ratio of 1:10 (m / v), along with 25 g of β-cyclodextrin. Perform ultrasonic extraction at 2000 W, 35℃, and 30 min for 30 min intervals, repeating the extraction 5 times. After extraction, pass the solution through a Buchner funnel and a 0.22 µm filter membrane to remove impurities, obtaining the ethyl acetate extract.

[0052] 3) Transfer the above ethyl acetate extract into a rotary evaporator at 40°C and 100 rpm. Then raise the temperature to 42°C and 120 rpm and evaporate for 30 min to remove most of the ethyl acetate.

[0053] 4) Continue to raise the temperature to 48°C and rotate the spindle at 150 rpm until the ethyl acetate is completely removed. After rotary evaporation, cool to room temperature to obtain the ethyl acetate extract of chrysanthemum and callus.

[0054] Experimental Example Component analysis of Huabai and callus extract The chemical components in the extracts of Columba salina and callus were characterized by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The main steps included sample processing, LC-MS detection, raw data acquisition, data preprocessing, data quality control, high-quality data acquisition, and qualitative analysis of metabolites.

[0055] Metabolomics analysis was conducted on the chemical components contained in extracts of *Hua Bai* and callus tissue, such as... Figure 1 This is the total ion chromatogram of chemical components in the extract of *Polygonum multiflorum* and callus tissue. The analysis results show that *Polygonum multiflorum* and callus tissue contain 2177 metabolites in 13 major categories, including flavonoids (12.13%), amino acids and their derivatives (10.75%), phenolic acids (10.47%), alkaloids (9.92%), terpenes (9.42%), and organic acids (4.09%). Figure 2 Among them, 46 bibenzyl derivatives and their derivatives were detected, including 3,3'-dihydroxy-2-p-hydroxybenzyl-5-methoxybibenzyl, 3,3',5-trihydroxybibenzyl, 3,3'-dihydroxy-5-methoxybibenzyl, yam extract III (3,3'-dihydroxy-5-methoxybibenzyl), dendrobine (3,3'-dihydroxy-5,4'-dimethoxybibenzyl), Dactylorhin E, etc.

[0056] Cytotoxicity test of Huabai and callus extract Human fibroblasts (HSF) were used to determine the cytotoxicity of samples containing different concentrations of extracts of *Wax glabra* and callus tissue according to Examples 1 and 4 using the MTT assay. The specific experimental steps are as follows: Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, the cells are seeded into 96-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0057] Experimental Groups: The experiment consisted of a zeroing group, a solvent control group (Control), a positive control group (PC), and a sample group. Within the sample group, each sample had eight concentration gradients, with three replicate wells for each concentration gradient.

[0058] Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 1).

[0059] Table 1 Test Concentration Setting Table

[0060] Drug administration: Drug administration was performed when the cell seeding rate in the 96-well plates reached 40%–60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of the sample was added to each well; for the zeroing group, no cells were seeded, only 200 μL of cell culture medium was added. After drug administration, the 96-well plates were placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0061] Detection: After culturing cells for 24 h, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The cells were incubated at 37 ℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. The OD value was read at 490 nm.

[0062] The formula for calculating relative cell viability is as follows: Relative cell viability (%) = (sample well OD - zeroing well OD) / (solvent control well OD - zeroing well OD) × 100%.

[0063] The test results of the extracts of Hua Bai and callus tissue prepared in Example 1 are shown in Table 2: Table 2. MTT assay results of extracts from Huabai and callus tissue prepared in Example 1.

[0064] Conclusion Analysis: The MTT test results show that the extracts of Huabai and callus prepared in Example 1 did not show significant cytotoxicity to human fibroblasts within the concentration range of 4%.

[0065] The test results of the Huabai and callus extracts prepared in Example 4 are shown in Table 3.

[0066] Table 3. MTT assay results of extracts from Huabai and callus tissue prepared in Example 4.

[0067] Conclusion Analysis: The MTT test results show that the extracts of Huabai and callus prepared in Example 4 did not show significant cytotoxicity to human fibroblasts within a concentration range of 1%.

[0068] Verification of the DNA repair efficacy of Huabai and callus extracts Gene CASP10 , PARP1 , BCL2L1 , GTF2H2B and GTF2H2C As core members of the apoptosis and DNA repair network, they play an indispensable role in maintaining skin homeostasis, resisting UV damage, and regulating damage repair. CASP10 (Caspase 10), as an initiating caspase, triggers the apoptosis cascade by activating downstream effector caspases, participating in the regulation of programmed cell death and immune regulation. PARP1 (Poly(ADP-ribose) polymerase 1) participates in DNA damage repair by modifying nucleoproteins with poly(ADP-ribose) ribosylation. BCL2L1 (Bcl-2-like protein 1), as an anti-apoptotic member of the Bcl-2 family, antagonizes apoptotic signals by inhibiting mitochondrial membrane permeability and preventing the release of cytochrome C; its downregulation can enhance the sensitivity of cells to apoptotic signals. GTF2H2B (General Transcription Factor IIH Subunit 2B) is a core member of the TFIIH complex. It participates in nucleotide excision repair (NER) by recognizing DNA damage sites, assisting in the unwinding of the DNA double strand and initiating repair, and is crucial for UV-induced DNA damage repair. GTF2H2C (General Transcription Factor IIH Subunit 2C) also belongs to the TFIIH complex. In NER, it stabilizes the binding of the complex to damaged DNA, promoting the excision of damaged fragments and subsequent DNA synthesis, thus maintaining genome stability.

[0069] The specific experimental steps are as follows: 1) The extracts of Johannesburg and callus prepared in Example 1 or Example 2 were diluted with DMEM medium to obtain DMEM culture medium containing 1% Johannesburg and callus extract.

[0070] 2) HFF cells (human fibroblasts) in logarithmic growth phase were subjected to a reaction at a concentration of 1*102 6Inoculated at a density of cells / well in 6-well culture plates and cultured at 37°C and 5% CO2 for 24 h.

[0071] 3) Discard the culture medium, wash twice with PBS, and except for the blank control group, the model group and sample group are given 7.5 J / cm³ of PBS. 2 Modeling was performed using UVA irradiation doses.

[0072] 4) After modeling, the cells were treated according to the model group and the sample group respectively (model group: add 2 mL of DMEM culture medium; sample group: add 2 mL of DMEM culture medium containing 1% Corydalis and callus extract). Each treatment was set up in 3 replicates and incubated in a 37℃, 5% CO2 culture environment for 24 h.

[0073] 5) After removing the cell culture medium and washing twice with PBS, cells were collected and total RNA was extracted. RNA purity and concentration were assessed, and a transcriptome library was constructed. The library was sequenced using a Llumina Novaseq 6000 sequencing platform. Reference genome alignment was performed using HISAT2 software, and gene expression levels (FPKM) were calculated to identify apoptosis-related genes between the sample group and the model group. CASP10 , PARP1 , BCL2L1 , GTF2H2B and GTF2H2C The fold change in gene expression levels (FC).

[0074] The results are as follows Figure 3 and Figure 4 As shown, the extracts of Corydalis rhamnoides and callus prepared in Example 1 can significantly promote the growth of apoptosis-related genes. CASP10 , PARP1 The expression of [certain substances] was upregulated by 177% and 15% respectively compared to the model group; while for [other substances]... BCL2L1 The expression of [genes] was not significantly promoted. The extracts of *Hua Bai* and callus prepared in Example 1 significantly promoted the expression of genes related to DNA mismatch repair, compared to the model group. GTF2H2B Gene expression was upregulated by 785%. GTF2H2C Gene expression was upregulated by 8379%.

[0075] Therefore, it can be seen that the extracts of chrysanthemum and callus obtained by this technical solution have a good effect on repairing skin cell DNA.

[0076] Verification shows that extracts of white chrysanthemum and callus tissue have the effect of repairing the skin barrier. The PIK3R1 and RELN genes influence skin barrier repair, immune regulation, and homeostasis by regulating the PI3K / AKT signaling pathway and extracellular matrix signaling, respectively. PIK3R1 (Phosphoinositide-3-Kinase Regulatory Subunit 1) is the p85α regulatory subunit of PI3K. Upon activation by the upstream receptor tyrosine kinase (RTK), the PIK3R1 protein recruits the PI3K complex to the cell membrane by binding to phosphorylated tyrosine residues of RTK, promoting the production of phosphatidylinositol-3,4,5-triphosphate (PIP3) from the catalytic subunit p110α, thereby activating the AKT / mTOR pathway and regulating cell growth, proliferation, and survival. RELN (Reelin, an extracellular matrix glycoprotein) activates the FAK / Syk / STAT3 and Akt / mTOR pathways by binding to integrin β1 and the receptors ApoER2 / VLDLR, enhancing tumor cell adhesion, proliferation, and glycolysis.

[0077] The specific experimental steps are the same as above. The relative expression levels of PIK3R1 and RELN, which are related to the PI3K-Akt signaling pathway, were calculated and analyzed in the blank control group, model group and sample group.

[0078] like Figure 5 As shown, the extracts of Huabai and callus prepared in Example 1 can promote the expression of PIK3R1 and RELN, which are related to the PI3K-Akt signaling pathway, and the expression levels are upregulated by 13% and 77%, respectively, compared with the model group.

[0079] CACNA1C (Calcium Voltage-Gated Channel Subunit Alpha1 C) acts as a transmembrane pore protein, regulating calcium ion influx. The calcium channel complex it forms (α1 / α2δ / β / γ subunits) participates in epidermal keratinocyte differentiation, intercellular junction formation, and skin barrier homeostasis by mediating calcium signaling. Abnormal CACNA1C function can lead to calcium gradient disturbances, affecting epidermal permeability barrier repair. CAMK4 (Calcium / Calmodulin-Dependent Protein Kinase IV) transduces calcium signaling into the nucleus through phosphorylation of transcription factors (such as CREB1 and MEF2D), regulating the expression of keratinocyte differentiation-related genes and participating in immune responses and inflammatory reactions. Its activity depends on changes in calcium ion concentration, playing a regulatory role in skin barrier function and damage repair.

[0080] The specific experimental steps are the same as above. The relative expression levels of cAMP signaling pathway-related genes CACNA1C and CAMK4 were calculated and analyzed in the blank control group, model group and sample group.

[0081] like Figure 6 As shown, the extracts of Codonopsis pilosula and callus prepared in Example 2 significantly promoted the expression of genes related to the cAMP signaling pathway. Compared with the model group, the expression level of CACNA1C gene was upregulated by 782%, and the expression level of CAMK4 gene was upregulated by 28%. Therefore, the Codonopsis pilosula and callus extracts obtained through this technical solution have the effect of repairing the skin barrier.

[0082] Verification of anti-aging effects of extracts from white chrysanthemum and callus tissue. Hepatocyte Growth Factor (HGF) activates downstream signaling pathways (such as MAPK / PI3K) by binding to c-Met receptors, regulating cell proliferation, migration, and morphogenesis. In skin injury repair, it promotes epidermal cell dedifferentiation and granulation tissue formation, accelerating wound healing. Fibroblast Growth Factor 1 (FGF1) activates the RAS-MAPK and PI3K-AKT pathways by binding to FGFR receptors, driving fibroblast proliferation, collagen synthesis, and angiogenesis. In skin regeneration, it promotes epidermal remodeling and extracellular matrix repair, and enhances barrier function by maintaining keratinocyte activity. Laminin subunit alpha-2 (LAMA2), a core component of the basement membrane, interacts with type IV collagen and nestin to form a three-dimensional network structure, maintaining the mechanical stability of the dermal-epidermal junction and promoting cell adhesion and migration during skin injury repair. COL4A5 (Collagen type IV alpha-5 chain), a major structural protein of the basement membrane, assembles with α3 and α4 chains to form a heterotrimer, creating a reticular scaffold to support epidermal cell anchorage. Mutations in COL4A5 can lead to abnormal collagen networks, weakening the tensile strength of the basement membrane and causing increased skin fragility and delayed dermal repair. The long isoform of COL18A1 (Collagen type XVIII alpha-1 chain) regulates angiogenesis and inflammatory responses through its C-terminal endostatin domain. It also synergistically enhances the stability of the basement membrane's dense layer with type IV collagen, promoting epidermal cell adhesion to the matrix. High expression of COL18A1 can improve skin barrier function and alleviate inflammatory damage in atopic dermatitis. COL14A1 (Collagen type XIV alpha-1 chain) belongs to the FACIT collagen family. It influences dermal mechanical properties by regulating the diameter and arrangement of type I collagen fibers. Upregulation of its expression enhances skin elasticity and resists UV-induced collagen degradation, while its deficiency leads to decreased skin tensile strength and abnormal scar repair. The SRC gene encodes a non-receptor tyrosine kinase, which regulates cell adhesion, migration, and proliferation by activating signaling pathways such as FAK and integrins. In skin wound repair, it promotes fibroblast migration and extracellular matrix remodeling, and simultaneously affects the stability of epidermal cell junctions by regulating the dynamics of the SRC-FAK complex.

[0083] The specific experimental steps are the same as above. The relative expression levels of HGF, FGF1, LAMA2, COL4A5, COL18A1, COL14A1 and SRC genes were calculated and analyzed in the blank control group, model group and sample group.

[0084] like Figure 7 As shown, the extracts of *Hypericum perforatum* and callus prepared in Example 2 promoted the expression of growth factor genes HGF and FGF1, with expression levels upregulated by 23% and 39%, respectively, compared to the model group. Figure 8 As shown, the extracts of *Hypericum perforatum* and callus prepared in Example 2 significantly promoted the expression of collagen synthesis-related genes LAMA2, COL4A5, COL18A1, and COL14A1, with upregulation of 17%, 27%, 856%, and 20%, respectively, compared to the model group. Figure 9 As shown, the extracts of Huabai and callus prepared in Example 2 can promote the expression of SRC, a gene related to cell proliferation, migration and adhesion, and its expression level is upregulated by 589% compared with the model group.

[0085] Verification shows that extracts of white chrysanthemum and callus tissue have stabilizing and anti-aging effects. LAMA2 (Laminin subunit alpha-2), a key component of the extracellular matrix (ECM), maintains skin structural integrity by mediating epidermal cell adhesion to the matrix and participates in post-injury barrier repair. ITGB1 (Integrin subunit beta-1) promotes cell migration and proliferation by regulating cell-ECM adhesion signaling; its upregulation can accelerate skin wound healing and enhance barrier regeneration. TJP1 (Tightjunction protein 1) is a core scaffold protein of the tight junction complex, ensuring barrier function by maintaining the stability of intercellular junction structures; abnormal expression or function can directly lead to increased skin barrier permeability. MMP3 (Matrix metalloproteinase 3) dynamically regulates barrier remodeling by degrading various ECM components such as laminin and collagen, balancing matrix metabolism processes in tissue repair and inflammatory responses.

[0086] The specific experimental steps are as follows: 1) The extracts of Johannesburg and callus prepared in Example 3 or Example 4 were diluted with DMEM medium to obtain DMEM culture medium containing 1% Johannesburg and callus extract.

[0087] 2) HFF cells (human fibroblasts) in logarithmic growth phase were subjected to a reaction at a concentration of 1*102 6 Inoculated at a density of cells / well in 6-well culture plates and cultured at 37°C and 5% CO2 for 24 h.

[0088] 3) After discarding the culture medium and washing twice with PBS, except for the blank control group, the model group and the sample group were given UVA irradiation dose of 7.5 J / cm2 for modeling treatment.

[0089] 4) After modeling, the cells were treated according to the model group and the sample group respectively (model group: add 2 mL of DMEM culture medium; sample group: add 2 mL of DMEM culture medium containing 1% Corydalis and callus extract). Each treatment was set up in 3 replicates and incubated in a 37℃, 5% CO2 culture environment for 24 h.

[0090] 5) After removing the cell culture medium and washing twice with PBS, cells were collected and total RNA was extracted. RNA purity and concentration were assessed, and a transcriptome library was constructed. The library was sequenced using a Llumina Novaseq 6000 sequencing platform. Reference genome alignment was performed using HISAT2 software to analyze gene expression levels and calculate the fold change (FC) of gene expression levels of skin barrier-related genes LAMA2, ITGB1, TJP1, and MMP3 between the sample group and the model group.

[0091] like Figure 10 As shown in Example 3, the extracts of *Hypericum perforatum* and callus prepared in this study promoted the expression of skin barrier-related genes LAMA2, ITGB1, and TJP1, with upregulation of 30%, 15%, and 76%, respectively, compared to the model group, thus promoting collagen regeneration and stabilizing the skin barrier. Simultaneously, it significantly inhibited MMP3 gene expression, with a 28% downregulation compared to the model group, reducing the degradation of extracellular matrix proteins. Therefore, the *Hypericum perforatum* and callus extracts obtained through this technique have the effects of maintaining skin structural stability and anti-aging.

[0092] Verification showed that extracts of Cosmodium and callus tissue have anti-inflammatory and soothing effects. MAPK13 (Mitogen-activated protein kinase 13), a member of the p38 MAPK family, mediates the inflammatory signaling cascade by activating downstream transcription factors ELK1 and ATF2 in response to pro-inflammatory cytokines or physical stress (such as ultraviolet radiation), and regulates the upregulation of pro-inflammatory factors such as CXCL14. GADD45G (Growtharrest and DNA damage-inducible gamma) activates the p38 / JNK pathway through MTK1 / MEKK4 kinases, inducing cell growth arrest under environmental stress (such as DNA damage or oxidative stress) and coordinating the transcriptional regulation of inflammation-related genes. Its expression level is closely related to the inflammatory damage repair process.

[0093] The specific experimental steps are the same as above. The relative expression levels of the cell inflammation-related genes MAPK13 and GADD45G were calculated and analyzed in the blank control group, model group and sample group.

[0094] like Figure 11 As shown, the extracts of *Polygonum multiflorum* and callus prepared in Example 3 can inhibit the expression of cellular inflammation-related genes MAPK13 and GADD45G. Compared with the model group, their expression levels were downregulated by 28% and 77%, respectively, inhibiting the production and release of pro-inflammatory factors. Therefore, the extracts of *Polygonum multiflorum* and callus obtained through this technical method have anti-inflammatory and soothing effects.

[0095] Verification showed that extracts of Cosmodium styracifolium and callus tissue have anti-photoaging effects. UNG (Uracil DNA glycosylase) is a base excision repair (BER) initiation enzyme that prevents UV-induced base mutations by specifically removing mis-incorporated uracil in DNA. Upregulation of UNG expression significantly enhances the skin cells' defense against UV-induced mutagenesis. TDG (Thymine DNA glycosylase) maintains genomic stability by excising thymine residues in G / T mismatches. Enhanced TDG activity effectively repairs UV-induced deamination damage, reducing the risk of skin cell carcinogenesis. MSH2 (MutS homolog 2), a core protein of DNA mismatch repair (MMR), recognizes and repairs replication errors and oxidative / alkylation damage. Upregulation of MSH2 synergistically enhances the skin's clearance efficiency of UV-induced mutagenic factors. PCNA (Proliferating cell nuclear antigen) recruits key BER enzymes (such as DNA polymerase β) to damage sites, promoting DNA gap filling and synthesis. Upregulation of PCNA expression accelerates UV damage repair and supports skin barrier regeneration. EXO1 (Exonuclease 1) performs 5'→3' excision of mismatched strands in the MMR, precisely removing UV-induced abnormal bases. Enhanced EXO1 activity improves the skin cell's genomic error correction capabilities. XPA (Xeroderma pigmentosum group A), as a core scaffold protein in NER, precisely removes large UV photoproducts such as CPD / 6-4PP in the tissue damage recognition complex, making it a key molecule in the skin's anti-photoaging mechanism. RPA1 (Replication protein A1) stabilizes replication forks by binding to single-stranded DNA and synergistically activates nucleotide excision repair (NER) with XPA; its acetylation modification specifically enhances the recognition and repair efficiency of UV damage. MNAT1 (Menage a trois 1) coordinates cell cycle and transcription-coupled repair by regulating CDK7 kinase activity; its participation in NER complex assembly enhances the skin's ability to clear UV-induced DNA adducts. POLE2 (DNA polymerase epsilon subunit 2) promotes replication-coupled repair by maintaining chromatin stability at the replication fork, and its high expression can synergistically enhance UV damage repair through the NER / BER pathway. POLE3 (DNA polymerase epsilon subunit 3) works synergistically with POLE2 to maintain replication fork nucleosome assembly, and promotes the proximity of NER proteins to UV damage sites through chromatin remodeling; its upregulation can enhance the tolerance of skin cells to persistent DNA damage.

[0096] The specific experimental steps are the same as above. The relative expression levels of DNA damage repair-related genes UNG, TDG, MSH2, PCNA, EXO1, XPA, RPA1, MNAT1, POLE2, and POLE3 were calculated and analyzed in the blank control group, model group, and sample group.

[0097] like Figure 12 As shown, the extracts of Cosmodium styracifolium and callus prepared in Example 4 can promote the expression of DNA damage repair-related genes UNG, TDG, MSH2, PCNA, EXO1, XPA, RPA1, MNAT1, POLE2, and POLE3. Compared with the model group, their expression levels were upregulated by 20%, 26%, 30%, 21%, 33%, 47%, 15%, 21%, 34%, and 21%, respectively, effectively improving the skin cells' defense against ultraviolet damage and enhancing repair efficiency. Therefore, the Cosmodium styracifolium and callus extracts obtained through this technical method have anti-photoaging and skin repair-accelerating effects.

[0098] The efficacy of extracts from gentian root bark and callus tissue in maintaining cell cycle balance was verified. CDK1 (Cyclin-dependent kinase 1), a member of the Ser / Thr protein kinase family, participates in cell cycle progression by regulating DNA replication initiation and cell-ECM adhesion complex function; its enhanced activity can promote skin cell proliferation and repair after DNA damage. CDK2 (Cyclin-dependent kinase 2) is a core regulator of the G1 / S phase transition, driving DNA synthesis initiation by binding to Cyclin E / A and activating the S-phase kinase complex. CCNB1 (Cyclin B1) forms a maturation-promoting factor with CDK1, specifically regulating the G2 / M phase transition and mitosis. PLK1 (Polo-like kinase 1) coordinates mitosis in the G2 / M phase by phosphorylating target proteins such as FOXO1; its expression deficiency leads to decreased cell viability and abnormal cell division. KNL1 (Kinetochore scaffold 1) plays a crucial role in maintaining the spindle assembly checkpoint function and precise chromosome segregation, thus contributing to the balance between cell proliferation and apoptosis. SKP2 (S-phase kinase-associated protein 2) mediates the degradation of p27Kip1 via the SCF ubiquitination complex, relieving its inhibition of CDK2 / Cyclin E and driving the S-phase progression. E2F2 (E2F transcription factor 2) is a core transcriptional regulator of the cell cycle, influencing the aging process by targeting genes related to apoptosis and DNA repair; its downregulation can induce premature aging phenotypes.

[0099] The specific experimental steps are the same as above. The relative expression levels and fold differences of cell growth cycle-related genes CDK1, CDK2, CCNB1, PLK1, KNL1, SKP2 and E2F2 between the sample group and the model group are calculated and analyzed.

[0100] The results are as follows Figure 13 As shown, the extracts of *Euphorbia lathyris* and callus prepared in Example 4 significantly promoted the expression of cell cycle-related genes CDK1 and CDK2, upregulating their expression levels by 55% and 28% respectively compared to the model group, effectively promoting skin cell proliferation and repair. They also promoted the expression of CCNB1, PLK1, KNL1, SKP2, and E2F2, upregulating their expression levels by 25%, 29%, 37%, 35%, and 413% respectively compared to the model group, helping to maintain cell cycle balance. Therefore, the extracts of *Euphorbia lathyris* and callus obtained through this technical method have the effect of maintaining cell cycle balance.

[0101] The study verified that extracts of Cosmodium styracifolium and callus tissue have moisturizing and soothing effects on human skin in vivo. Referring to T / CAB 0152—2022 "Test Methods for Seven Efficacy Items of Cosmetics: Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing and Soothing", the CK multifunctional skin tester was used to measure the changes in human transepidermal water loss value (skin TEWL value) and skin hemoglobin content (E value) to analyze and verify the moisturizing and soothing skin care effects of the Huabai and callus tissue extracts prepared in Example 1.

[0102] The specific testing process for human efficacy testing is as follows: Test Groups: Blank group: After cleansing, no test sample was used; Control group: After cleansing, the reagent (pure water) for dissolving the sample was evenly applied; Experimental group: After cleansing, the test sample (1% Wah Blanc and callus extract sample) was evenly applied.

[0103] Subject Recruitment: 30 qualified volunteers were recruited according to the requirements and signed written informed consent forms. Before enrollment, subjects were asked a series of questions regarding their medical history and health status based on the inclusion and exclusion criteria. Skin conformity assessments were also conducted and recorded.

[0104] Preparation before testing: After cleansing their face, the subjects sat quietly in a constant temperature and humidity room for 30 minutes; the instrument was tested before use.

[0105] Tape peeling-induced skin damage: Apply hypoallergenic tape to the test area, press for 2 seconds and then slowly peel it off, continuing to peel until visible redness appears.

[0106] Efficacy test: Subjects underwent instrumental evaluation of skin indicators (skin TEWL value, skin hemoglobin content (E value)) before application (T0), 1 h after application (T1), 4 h after application (T2), and 8 h after application (T3).

[0107] Statistical software was used to perform a normality test on the obtained data. One-way ANOVA was used to determine if the data conformed to a normal distribution; otherwise, a nonparametric test—the Wilcoxon signed-rank test—was used. Here, "ns" indicates P > 0.05, meaning no significant difference; "*" indicates P < 0.05, meaning a significant difference; and "**" indicates P < 0.01, meaning a highly significant difference.

[0108] Test Results: Skin TEWL values ​​were assessed in 30 subjects before and after using 1% chlorophyll and callus extract samples (Table 4 and 1 h and 4 h after use). Figure 14 The lower the skin's TEWL value, the less moisture is lost from the skin.

[0109] Table 4. Results of skin TEVL value testing after using 1% Huabai and callus extract samples.

[0110] As shown in Table 4, compared with the control group, the skin TEWL value of the subjects' evaluation area was significantly reduced by 17.00% (P<0.01) 1 h after using the 1% Huabai and callus extract sample; the skin TEWL value of the subjects' evaluation area was significantly reduced by 18.07% (P<0.01) 4 h after using the 1% Huabai and callus extract sample; and the skin TEWL value of the subjects' evaluation area was significantly reduced by 19.38% (P<0.01) 8 h after applying the sample. Compared with the control group, there was no significant difference in skin TEWL value after the subjects used the control group (pure water) for 1 h, 4 h, and 8 h.

[0111] like Figure 14 As shown in the figure, compared with the control group, the difference in skin TEWL values ​​between the subjects and the pre-treatment values ​​decreased by 9.0-fold (P<0.01), 38.1-fold (P<0.01), and 2.4-fold (P<0.01) respectively after 1 h, 4 h, and 8 h of using the 1% Huabai and callus extract sample. Therefore, the improvement in skin TEWL values ​​after 1 h, 4 h, and 8 h of using the 1% Huabai and callus extract sample was significantly better than that in the control group.

[0112] Test Results: In 30 subjects, skin hemoglobin content (E value) was assessed using instruments before and 1 h, 4 h, and 8 h after using 1% Corydalis and callus extract samples (Table 5 and...). Figure 15 The lower the E value, the lower the skin hemoglobin content.

[0113] Table 5. Results of skin hemoglobin level test after using 1% Corydalis and callus extract samples.

[0114] As shown in Table 5, compared with the control group, after 1 h of application of 1% Huabai and callus extract, the skin heme content (E value) in the evaluation area of ​​the subjects was significantly reduced by 9.57% (P < 0.05); after 4 h of application of 1% Huabai and callus extract, the skin heme content (E value) in the evaluation area of ​​the subjects was significantly reduced by 14.72% (P < 0.01); and after 8 h of application, the skin heme content (E value) in the evaluation area of ​​the subjects was significantly reduced by 15.99% (P < 0.01). Compared with the control group, there was no significant difference in skin heme content (E value) after 1 h, 4 h, and 8 h of application of pure water.

[0115] like Figure 15 As shown in the figure. Compared with the control group, after using 1% *Huabella salina* and callus extract for 1 h, 4 h, and 8 h, the difference in skin hemoglobin levels between the subjects and the pre-treatment values ​​decreased by 18.3 times (P<0.01), 26.6 times (P<0.01), and 34.0 times (P<0.01), respectively. Therefore, the improvement in skin hemoglobin levels after using 1% *Huabella salina* and callus extract for 1 h, 4 h, and 8 h was significantly better than that in the control group. Human efficacy testing results showed that 1% *Huabella salina* and callus extract can significantly reduce skin moisture loss and lower skin hemoglobin content, exhibiting moisturizing and soothing skincare effects.

[0116] As can be seen from the above embodiments, the present invention provides a Huabai and callus extract with anti-aging, skin barrier repair, moisturizing, soothing, anti-photoaging, DNA repair, and cell cycle balancing effects. Bibenzyls are rarely found and used in plant extracts. Existing research mainly focuses on Dendrobium. Based on published data on the types of bibenzyls in 46 Dendrobium species, except for Dendrobium officinale which contains 36 types of bibenzyls, the other Dendrobium species contain no more than 16 types of bibenzyls, and most contain only a few (see He L, Su Q, Bai L, et al. Recent Research Progress on Natural Small Molecule Bibenzyls and its Derivatives in Dendrobium species[J]. European Journal of Medicinal Chemistry, 2020). (204:112530.DOI:10.1016 / j.ejmech.2020.112530.), Compared with Dendrobium, Huabai and callus extract contain more (46 kinds) of bibenzyl compounds; compared with general plant extracts, Huabai and callus extract have more comprehensive effects, and when applied to cosmetics, they can achieve more effects at once.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Huabai and callus extracts in the preparation of anti-aging products.

2. Application of Huabai and callus extracts in the preparation of skin barrier repair products.

3. Application of Huabai and callus extracts in the preparation of moisturizing products.

4. Application of Huabai and callus extracts in the preparation of soothing products.

5. Application of Huabai and callus extracts in the preparation of anti-photoaging products.

6. Application of Huabai and callus extracts in the preparation of DNA repair products.

7. Application of Huabai and callus extracts in the preparation of balanced cell cycle products.

8. The application according to any one of claims 1 to 7, characterized in that, The product in question is a cosmetic product.

9. The application according to any one of claims 1 to 7, characterized in that, The preparation method of the Huabai and callus extract includes the following steps: The callus tissue and the white chrysanthemum were mixed with an alcohol reagent and subjected to alcohol extraction to obtain the alcohol extract. The alcohol extract was subjected to rotary evaporation to obtain the rotary evaporation product; The rotary evaporation product was subjected to enzymatic hydrolysis to obtain the enzymatic hydrolysis product, which is the extract of Hua Bai and callus tissue. The alcohol reagent is ethanol, and the volume percentage concentration of the ethanol is 50-99%. The weight ratio of the white chrysanthemum and callus tissue to the volume ratio of the alcohol reagent is 1g:3~8L; The alcohol extraction process is accompanied by an ultrasonic environment, the power of which is 2000~3000W. The alcohol extraction process is repeated 3 to 8 times, with each extraction lasting 20 to 100 minutes and an interval of 20 to 40 minutes. The temperature for the alcohol extraction treatment is 20~40℃; The alcohol extract is further filtered sequentially through a Buchner funnel and a 0.22 µm filter membrane before being subjected to rotary evaporation. The rotary evaporation process includes first processing at 30~40℃ and 50~150rpm, and then processing at 40~60℃ and 100~200rpm. The enzyme reagent used in the enzymatic hydrolysis treatment is protease; The enzymatic hydrolysis process includes the following steps: The rotary evaporation product was mixed with water and protease, incubated, and then subjected to enzyme inactivation treatment. The volume ratio of the rotary evaporation product to water to the weight of the protease is 1~3:20~40g; The incubation temperature is 40~50℃, and the time is 1~3h; The enzyme inactivation treatment is carried out at 80-90°C for 10-20 minutes. And / or, the enzymatic hydrolysis product further includes filtration through a 0.22 µm filter membrane; The enzymatic hydrolysis products also undergo freeze-drying treatment; Before the Huabai and callus tissue are mixed with the alcohol reagent, they undergo a mixed liquid nitrogen grinding process. The Huabai and callus tissue are Huabai and callus tissue dry powder; The target particle size for the mixed liquid nitrogen grinding treatment is ≤40 mesh.

10. The application according to any one of claims 1 to 7, characterized in that, The preparation method of the Huabai and callus extract includes the following steps: The callus tissue, ethyl acetate, and β-cyclodextrin were mixed and extracted with ethyl acetate to obtain the ethyl acetate extract. The ethyl acetate extract was subjected to rotary evaporation to obtain the rotary evaporation product, which is the extract of Huabai and callus tissue. The weight ratio of the white chrysanthemum and callus tissue to the volume ratio of ethyl acetate is 1g:8~12L; The weight ratio of the white fungus and callus tissue to β-cyclodextrin is 10~30:1; The ethyl acetate extraction process is performed under an ultrasonic environment with a power of 1500~3000W. The ethyl acetate extraction process is performed 3 to 8 times, with each extraction lasting 20 to 100 minutes and an interval of 20 to 40 minutes. The temperature for the ethyl acetate extraction treatment is 20~40℃; The ethyl acetate extract is further filtered sequentially through a Buchner funnel and a 0.22 µm filter membrane before being subjected to rotary evaporation. The rotary evaporation process includes first processing at 30~40℃ and 80~110rpm, then processing at 40~45℃ and 110~130rpm, and finally processing at 45~50℃ and 130~180rpm. Before the Huabai and callus tissue are mixed with the alcohol reagent, they undergo a mixed liquid nitrogen grinding process. The Huabai and callus tissue are Huabai and callus tissue dry powder; The target particle size for the mixed liquid nitrogen grinding treatment is ≤40 mesh.

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