Saussurea obtusifolia callus extract as well as preparation method and application thereof

Abstract

The invention relates to an application of a saussurea obtusifolia callus extract in preparation of a skin external preparation, a beautifying oral preparation, a health care product, food or a food additive. The saussurea obtusifolia callus extract has excellent moisturizing, soothing and repairing effects, also has multiple effects of resisting aging, whitening, resisting wrinkles, tightening, removing acnes, resisting oxidation, preventing hair loss, protecting hair, improving sleep, enhancing immunity, improving autophagy ability and the like, and can be applied to skin and hair care products and health-care foods as an active additive. The saussurea obtusifolia callus cells are used as raw materials, so that the method has the advantages of infinite proliferation, stable quality among batches, short culture period, environmental friendliness and easiness in batch culture in unrelated seasons, can be produced under the condition of not damaging plants, and has great value and significance for sustainable development and utilization of plant resources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of daily products, and particularly relates to a Saussurea obvallata callus extract and a preparation method and application thereof. BACKGROUND

[0002] Saussurea obvallata (DC.) Sch.-Bip. is a plant of the Asteraceae family and the Saussurea genus, with a plant height of 16-60 cm, an erect stem, and short pubescence or no pubescence. It grows at an altitude of 3200-4700 m and is a plant unique to highland areas. The Saussurea obvallata is different from the present market popular Saussurea involucrata, with blue-purple flowers and long-circular achenes, and is mainly distributed in Gansu, Qinghai, Tibet, Sichuan, Yunnan and other places. The Saussurea obvallata is warm and slightly bitter, and has the effects of clearing heat and resolving toxins, dispelling wind and dampness, and dredging channels and activating blood. In Tibetan medicine, it is used for treating rheumatic arthritis, high-altitude discomfort, irregular menstruation, and retained placenta. At present, the Saussurea obvallata is still in a wild state, and no one has successfully planted it. Therefore, there are few reports on active ingredients, pharmacological activities, and callus culture of the Saussurea obvallata, and there is no report on the research and application of the Saussurea obvallata and callus in skin care. SUMMARY

[0003] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a Saussurea obvallata callus extract and a preparation method and application thereof.

[0004] One aspect of the present application provides a preparation method of a Saussurea obvallata callus extract, wherein the method comprises the following steps: a) culture of callus cells: The tender buds of the Saussurea obvallata are inoculated into an induction medium MS + 6-BA 0.9 ~ 1.1 mg / L + NAA 1.9 ~ 2.1 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum to induce callus, The callus cells are inoculated into a proliferation medium MS + 6-BA 1.3 ~ 1.7 mg / L + NAA 1.8 ~ 2.2 mg / L + KT 1.8 ~ 2.2 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum, and the volume of the inoculation proliferation medium is 1% ~ 2% of the callus cells are proliferated in the callus proliferation medium to obtain the Saussurea obvallata callus cells; Further, the induction medium is MS + 6-BA 1.0 mg / L + NAA 2.0 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum. The proliferation medium is MS + 6-BA 1.5 mg / L + NAA 2.0 mg / L + KT 2.0 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum.

[0005] b) Preparation of the extract of the E. przewalskii Reichb. var. glabra Reichb. callus: The E. przewalskii Reichb. var. glabra Reichb. callus cells obtained after the value-added cultivation are added into water for extraction to obtain the extract of the E. przewalskii Reichb. var. glabra Reichb. callus.

[0006] Further, the callus culture conditions are: temperature 20±1℃, humidity 50%~70%, Dark culture.

[0007] The E. przewalskii Reichb. var. glabra Reichb. callus cells are induced using the induction medium, and the induction rate can reach 52.50%. When the value-added medium is used for value-added culture, the callus does not have problems such as browning or water spotting, and the proliferation coefficient can reach 1.6 times after 30 days of culture, and the value-added rate is relatively optimal.

[0008] Further, the extraction of the E. przewalskii Reichb. var. glabra Reichb. callus cells comprises the following steps: A. The E. przewalskii Reichb. var. glabra Reichb. callus cells are weighed and ground; B. 10 ~ times the volume of deionized water, preferably 20 times the volume of deionized water, is added for immersion stirring; C. 550 ~ W, preferably 600 W, is used for ultrasonic extraction, centrifugal filtration, and the supernatant is collected for filtration, and the filtrate is combined; D. Drying is performed to obtain the extract of the E. przewalskii Reichb. var. glabra Reichb. callus.

[0009] Another aspect of the present application provides an extract of the E. przewalskii Reichb. var. glabra Reichb. callus obtained by the above preparation method, wherein the total polyphenol content in the dry weight of the E. przewalskii Reichb. var. glabra Reichb. callus is 35-45 mg / g, the flavonoid content is 55-65 mg / g, and the protein content is 535-545 mg / g.

[0010] Further, the total polyphenol content in the dry weight of the E. przewalskii Reichb. var. glabra Reichb. callus is 40.34 mg / g, the flavonoid content is 59.42 mg / g, and the protein content is 541.93 mg / g.

[0011] The application also provides an application of a rosette snow lotus callus extract in preparation of a skin external agent, a hair-care external agent, a beauty oral agent, a health-care product, a food or a food additive for moisturizing, soothing and repairing, anti-aging, whitening, tightening and anti-wrinkling, acne removal, anti-oxidation, hair loss prevention and scalp care, sleep improvement, immunity enhancement and autophagy capacity improvement.

[0012] Further, the moisturizing effect is to promote aqp3a gene expression and hyaluronic acid synthesis; the soothing and repairing effect is to repair skin ultraviolet damage, promote ATP content increase, DNA damage repair, and inhibit TNF-α, IL-1α, IL-6 and IL-8 inflammatory factor expression; the anti-aging effect is to promote human dermal fibroblast proliferation, inhibit β-galactosidase activity, promote telomerase activity, increase superoxide dismutase (SOD) activity and increase glutathione (GSH) content; the whitening effect is to inhibit tyrosinase activity and melanin production of human epidermal melanocyte B16; the tightening and anti-wrinkling effect is to increase elastin eln2 gene expression and col1a1a gene expression; the acne removal effect is to reduce neutrophil content; the sleep improvement effect is to promote expression of melatonin receptor gene (mtnr1aa) and / or GABA receptor gene (GABRB3); the immunity enhancement effect is to increase macrophage activity; the hair loss prevention and scalp care effect is to enhance blood microcirculation; and the autophagy capacity improvement effect is to increase LC3-Ⅱ protein expression. gabra1 )expression; the immunity enhancement effect is to increase macrophage activity; the hair loss prevention and scalp care effect is to enhance blood microcirculation; the autophagy capacity improvement effect is to increase LC3-Ⅱ protein expression.

[0013] The application also provides a skin external agent, wherein the content of the rosette snow lotus callus extract in the skin external agent is 0.001% to 1%, and the percentage refers to the mass percentage of the rosette snow lotus callus extract in the total mass of the skin external agent; preferably, the content of the rosette snow lotus callus extract is 0.01% to 1%.

[0014] The application also provides a beauty oral agent, wherein the content of the rosette snow lotus callus extract in the beauty oral agent is 0.001% to 1%, and the percentage refers to the mass percentage of the rosette snow lotus callus extract in the total mass of the beauty oral agent; preferably, the content of the rosette snow lotus callus extract is 0.01% to 1%.

[0015] The application also provides a health-care product, wherein the content of the rosette snow lotus callus extract in the health-care product is 0.001% to 1%, and the percentage refers to the mass percentage of the rosette snow lotus callus extract in the total mass of the health-care product; preferably, the content of the rosette snow lotus callus extract is 0.01% to 1%.

[0016] Advantages The present application obtains the calla snow lotus callus by using the method of plant cell tissue culture, and obtains the calla snow lotus callus extract after extraction. In the induction culture, the induction rate reaches 52.50%, the water staining and browning do not occur in the subculture, the subculture speed is faster, and a large amount of calla snow lotus callus extract can be efficiently obtained.

[0017] The calla snow lotus callus extract prepared in the present application contains high active ingredients such as polyphenols, proteins and flavonoids, and has excellent moisturizing, soothing, repairing effects, and also has multiple effects such as anti-aging, whitening, anti-wrinkle tightening, acne removal, antioxidant, hair loss prevention, improvement of microcirculation, improvement of sleep, enhancement of immunity, etc., and can be applied as an active additive in skin care and hair care products and health foods.

[0018] The calla snow lotus callus extract obtained in the present application has the advantages of green environmental protection, stable quality and excellent effect, and solves the problems of wild plant resources and difficult development and utilization of calla snow lotus. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A is a wild plant of calla snow lotus, B is calla snow lotus medicinal material, and C is calla snow lotus callus Figure 2 Example two 2 (d) Table 7 Comparison of DNA fluorescence intensity of calla snow lotus callus extract and control group DETAILED DESCRIPTION

[0020] The present application will be described in detail below in combination with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be pointed out that those skilled in the art can make several changes and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0021] The experimental methods not specified in the following examples are selected according to conventional methods and conditions, or according to the instructions of the commodity. The reagents and raw materials used in the present application are commercially available.

[0022] Example one Culture of callus and preparation of callus extract 1. Culture method of calla snow lotus callus 1.1 Preparation of culture medium and culture conditions Seed sterilization and aseptic seedling culture are both MS basic medium. The proliferation medium is selected from MS medium with the addition of plant growth regulators. The above medium is added with 30 g / L sucrose, 3.5 g / L gellan gum, adjusted to pH 5.7-5.8, and sterilized in a sterilization pot at 121°C for 20 min for standby. The daily culture conditions are temperature 20±1°C, humidity 50%-70%, and dark culture.

[0023] The basic medium MS (M519) and plant growth regulators are purchased from Pytotechnolagy Company. The plant growth regulators include: naphthalene acetic acid (NAA); 6-benzylaminopurine (6-BA); 6- glycosylaminopurine (KT).

[0024] 1.2 Disinfection of explants and culture of aseptic seedlings After pruning, the seeds of wild Ligularia sibirica are soaked in gibberellin at room temperature for 24 h, washed under running water for 20 min, and then washed with sterile water for 5 times. Then, in a sterile clean bench, the seeds are sterilized with 75% ethanol (with Tween 20) for 15 seconds, washed with sterile water for 5 times, treated with 2% (V / V) NaClO solution (with Tween 20) in a shaker at 110 rpm for 10 min, washed with sterile water for 5 times, and then placed on MS basic medium at 20°C and light / dark for 12 h. After the seeds germinate and grow to about 1 cm, the germination rate is counted, and the seeds are transferred to MS culture bottles for aseptic seedling culture.

[0025] 1.3. Induction and proliferation of callus In a sterile clean bench, the leaves of aseptic seedlings are cut into segments with a length of 0.5-1.0 cm with a scalpel, inoculated into the induction medium: MS + 6-BA 1.0 mg / L + NAA 2.0 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum, and the induced callus is cultured in the proliferation medium: MS + 6-BA 1.5 mg / L + NAA 2.0 mg / L + KT 2.0 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum. When cultured in the induction medium, the induction rate can reach 52.50%, and when cultured in the proliferation medium, there is no problem such as browning or water spotting, the proliferation coefficient can reach 1.6 times after 30 days of culture, and a large amount of callus is obtained.

[0026] 2. Preparation of Ligularia sibirica extract and detection of active ingredients 2.1 Preparation of extract Take the callus of Saussurea involucrata, the plant of Saussurea involucrata and the plant of Saussurea involucrata var. decurrens, respectively, crush them, add water (1:10), extract them by ultrasonic (600W) for 30min, filter the filtrate, and obtain the extract of callus of Saussurea involucrata, the extract of plant of Saussurea involucrata and the extract of plant of Saussurea involucrata var. decurrens, respectively.

[0027] 2.2 Determination of total flavonoids content Standard solution: prepare a quercetin solution of 0.2315 mg / mL in ethanol, and dilute it to 2-10 times of the concentration gradient. Take 300 μL of sample solution, add 50 μL of 5% NaNO2, mix, and let it stand at room temperature for 6 min; then add 50 μL of 10% Al(NO3)3, mix, and let it stand at room temperature for 6 min; finally, add 500 μL of 4% NaOH and 140 μL of water, mix, and let it stand at room temperature for 15 min. Take 100 μL of the mixture to determine the OD value at 500 nm, and calculate the total flavonoids content in the sample according to the standard curve. The results are shown in Table 1: the total flavonoids content in the callus of Saussurea involucrata, the plant of Saussurea involucrata and the plant of Saussurea involucrata var. decurrens is 59.42 mg / g dry weight, 21.09 mg / g dry weight and 41.88 mg / g dry weight, respectively.

[0028] 2.3 Determination of total polyphenols content Standard curve: dilute the gallic acid solution of 0.33 mg / mL to 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, 0.3300 mg / mL concentration gradient. Take 100 μL of sample solution, add 500 μL of 10% Folin-Ciocalteu reagent, mix well, and let it stand for 5 min (not more than 8 min), then add 400 μL of 7.5% Na2CO3, mix well, and let it stand at room temperature for 60 min. Finally, take 100 μL to determine the OD value at 765 nm on the microplate reader, and calculate the total polyphenols content in the sample according to the standard curve. The results are shown in Table 1: the total polyphenols content in the callus of Saussurea involucrata, the plant of Saussurea involucrata and the plant of Saussurea involucrata var. decurrens is 40.34 mg / g dry weight, 9.15 mg / g dry weight and 18.85 mg / g dry weight, respectively.

[0029] 2.4 Determination of protein content The protein content was determined by BCA protein concentration assay kit: 20 μL of sample was taken, 200 μL of BCA working solution was added, and the absorbance was detected at 562 nm after incubation at 37ºC for 20-30 minutes. The protein content in the sample was calculated according to the BSA standard solution curve. The results are shown in Table 1. The total polyphenol content in the bud-leaved saussurea callus, bud-leaved saussurea plant and tianshan saussurea plant was 541.93 mg / g dry weight, 277.25 mg / g dry weight and 90.77 mg / g dry weight, respectively.

[0030] Table 1. Active ingredient content of saussurea extract Project name Total polyphenol content (mg / g) Total flavonoid content (mg / g) Protein content (mg / g) Bract saussurea callus 40.34 59.42 541.93 Bract saussurea plant 9.15 21.09 277.25 Tianshan saussurea plant 18.85 41.88 90.77 In summary, the active ingredients in bud-leaved saussurea can be effectively enriched by plant tissue culture process. The total polyphenol, total flavonoid and protein content in bud-leaved saussurea callus are higher than those in the medicinal material extract, and also better than those in tianshan saussurea extract.

[0031] 1 mL of bud-leaved saussurea callus extract was taken and analyzed by LC-MS to identify the active ingredients, such as syringin and glycyurarin.

[0032] Example 2: Efficacy test of bud-leaved saussurea callus extract 1. Moisturizing effect of bud-leaved saussurea callus extract (a) Effect of bud-leaved saussurea callus extract on aqp3a Relative expression of genes Zebrafish has high similarity in physiology, development and metabolism with mammals, and can reliably simulate human physiological and pathological processes. It has high homology with human genes, up to 87%, and its skin structure and function are very similar to those of humans. More and more people at home and abroad use zebrafish model to evaluate the efficacy of cosmetics and their raw materials.

[0033] Zebrafish is treated with sodium chloride, and the skin surface will shrink due to osmotic pressure. Hyaluronic acid (HA) exists in the dermis of the skin and has a moisturizing function. Aquaporin (AQP) is a protein located on the cell membrane, which forms a "pore" on the cell membrane and can control the entry and exit of water in cells. AQP-3, i.e. aquaporin 3, is mainly the epidermal aquaporin, which is a membrane transporter of water and glycerol, expressed in the plasma membrane of keratinocytes in the basal layer of the epidermis of normal skin, promoting water permeability and hydration of the stratum corneum. Therefore, by detecting aqp3a the relative expression of genes, it can be shown whether the sample has moisturizing efficacy.

[0034] Randomly select zebrafish in 6-well plates, 30 per well. Water-soluble administration of 0.05% of the bud leaf saussurea involucrata callus extract sample, while setting up normal control group and model control group, 3 mL per well. Three biological repeats. At the same time, water-soluble administration of sodium chloride to establish a zebrafish skin dehydration model. Incubate in the dark at 28°C for 22 h. Extract total RNA from each experimental group of zebrafish, synthesize cDNA, and use q-PCR to detect β-actin gene expression. Use β-actin as the internal reference for gene expression, calculate the relative expression of the target gene.

[0035] Table 2. Effect of bud leaf saussurea involucrata callus extract on aqp3a gene relative expression content Project name Detection concentration Gene relative expression Normal control group - 2.71 Model control group (sodium chloride stimulation) - 1 Bract saussurea callus extract 0.05% 2.24 The results show that compared with the model control group, the sample bud leaf saussurea involucrata callus extract aqp3a gene relative expression content increased by 2.24 times, close to the normal control group, indicating that the sample has strong moisturizing effect.

[0036] (b) Effect of bud leaf saussurea involucrata callus extract on hyaluronic acid content Hyaluronic acid (HA) exists in the extracellular matrix between cells and is a filler between cells, with moisturizing function. Both epidermis and dermis contain hyaluronic acid. When the skin is long-term dehydrated and dry, the water content of skin tissue cells and cells decreases, the hyaluronic acid content in the skin decreases, and the space filled by the gel matrix composed mainly of hyaluronic acid between cells decreases, resulting in close arrangement of cells, dehydration and hardening of collagen, causing aging of the stratum corneum, making the skin rough, dry, and losing elasticity. Zebrafish epidermis has a certain osmotic pressure tolerance range, and beyond the tolerance range will cause dehydration, resulting in skin surface shrinkage and reduced hyaluronic acid content in the body. Therefore, by detecting the content of hyaluronic acid, it can be shown whether the sample has moisturizing effect.

[0037] Randomly select zebrafish in 6-well plates, 30 per well. Water-soluble administration of the sample, while setting up normal control group and model control group, 3 mL per well. Three biological repeats. Except for the normal control group, water-soluble administration of sodium chloride to establish a zebrafish skin dehydration model. Incubate in the dark at 28°C for 22 h. After the experiment, collect the zebrafish samples of each group, grind and homogenize, centrifuge to obtain the supernatant, use the zebrafish hyaluronic acid (HA) quantitative detection kit, use the enzyme-labeled instrument to collect data, analyze the hyaluronic acid content (C) in zebrafish, calculate and judge whether the sample has moisturizing effect according to the formula, the calculation formula is as follows:

[0038] Table 3. Effect of Saussurea obvallata callus extract on the efficacy of hyaluronic acid Project name Detection concentration Hyaluronic acid content (ng / mL) Moisturizing efficacy (%) Normal control group - 215 Model control group (sodium chloride stimulation) - 178 Bract saussurea callus extract 0.05% 223 122 As shown in Table 3, compared with the model control group, the sample Saussurea obvallata callus extract can significantly increase the content of hyaluronic acid in zebrafish, and its moisturizing efficacy can reach 122% (P < 0.001), which has a certain moisturizing effect. p <0.001), which has a certain moisturizing effect.

[0039] In summary, the Saussurea obvallata callus extract with a mass percentage of 0.05% can promote the expression of aqp3a genes, thereby promoting the water permeability of the epidermis and the water content of the stratum corneum, and at the same time increasing the content of hyaluronic acid in the skin, which reflects its moisturizing efficacy.

[0040] 2. Soothing and repairing effect of Saussurea obvallata callus extract (a) Repairing effect of Saussurea obvallata callus extract on UV-damaged cells Skin aging is divided into endogenous aging and exogenous aging, and sunlight, especially ultraviolet (UV) irradiation, is the main factor for the formation of exogenous aging, so exogenous aging is also called photoaging. The main factors related to skin photoaging are UVA (320-400 nm) and UVB (280-320 nm). Many data show that UV can induce apoptosis in fibroblasts, mitochondrial DNA deletion changes, and an increase in intracellular reactive oxygen free radicals, so we use UVB irradiation of human fibroblasts (FB) to establish a skin photo-damage model, and use this model to evaluate the repairing and anti-photoaging effects of the sample.

[0041] Human fibroblasts (FB) were seeded in a 96-well plate at a cell density of 6000 cells / well and cultured in a 37°C 5% CO2 cell incubator for 24 hours. Then the FB cells were cultured in medium containing 0.05% and 0.005% Saussurea obvallata callus extract and Saussurea obvallata plant extract samples for 24 hours. After the medium was replaced with PBS, UVB irradiation was performed at a dose of 30 mJ / cm 2 . Then the medium containing 0.05% and 0.005% Saussurea obvallata callus extract and Saussurea obvallata plant extract samples was added, and the cells were cultured for another 24 hours. Cell viability was detected by CCK8 method. The untreated group was used as the blank control group, and the UVB irradiation group without sample was used as the model control group.

[0042] The results are shown in Table 4, Table 4. Protective effect of Saussurea obvallata callus extract on UV radiation damage of FB cells

[0043] From Table 4, it can be seen that after UVB irradiation, the cell viability of FB cells was significantly reduced to 69%; the extract of the callus of B. royleana at a concentration of 0.005% and 0.05% could significantly improve the cell viability of FB cells; and at the same concentration, the extract of the callus of B. royleana was better than the extract of B. royleana plant. In summary, it is shown that the extract of the callus of B. royleana has a better effect of repairing and resisting photoaging.

[0044] (b) Effect of the extract of the callus of B. royleana on the total movement distance of zebrafish Glacial acetic acid has strong penetrability and corrosiveness. The skin will feel a sharp pain and burning sensation when it comes into contact with glacial acetic acid. Zebrafish with incomplete development of swimming ability are selected, and glacial acetic acid is used to stimulate zebrafish to cause a larger area and a longer time of inflammatory pain, resulting in more movement trajectories and more severe pain. The total movement distance of zebrafish is detected by a behavior analyzer to evaluate whether the sample has a soothing effect.

[0045] Zebrafish were randomly selected in a 6-well plate, one fish per well. The sample of the extract of the callus of B. royleana was given at a weight ratio of 0.05% in water, and a normal control group and a model control group were set up, with a volume of 3 mL per well. Incubate at 28°C in the dark for 24 h. Ten zebrafish were randomly selected from each experimental group and transferred to a 96-well plate, and a glacial acetic acid solution was given to establish a zebrafish pain model. The movement trajectory of zebrafish was measured by a behavior analyzer, and the total movement distance (D) of zebrafish was analyzed and collected. Whether the sample has a soothing effect is calculated and judged according to the formula.

[0046]

[0047] The detection results are shown in Table 5: Table 5. Effect of the extract of the callus of B. royleana on the total movement distance of zebrafish Project name Detection concentration Total movement distance (mm) Relieving efficacy (%) Normal control group - 115 Model control group (glacial acetic acid stimulation) - 5662 Bract saussurea callus extract 0.05% 4536 20 As shown by the results in Table 5, compared with the model group, the extract of the callus of B. royleana at a concentration of 0.05% can reduce the pain caused by the stimulation of glacial acetic acid, and significantly reduce the total movement distance of zebrafish, revealing that the sample has a soothing effect.

[0048] (c) Effect of the extract of the callus of B. royleana on the relative expression amount of pro-inflammatory cytokine genes After sodium lauryl sulfate (SLS) acts on the body, it can trigger a stimulation reaction of the body. The stimulant enters the body of zebrafish, induces an inflammatory response, and the neutrophils have an immune response, migrate and gather to the skin epidermis. TNF- αis the earliest and most important inflammatory cytokine in the inflammatory response process, which can activate neutrophils; IL-1 plays a key role in transmitting information, activating and regulating immune cells, proliferation and differentiation of inflammation in the inflammatory response. IL-1 has two different molecular forms, one is called IL-1 α , and the other is called IL-1 β ; IL-6 can promote the up-regulation of neutrophil function, promote and regulate the secretion of inflammatory mediators such as cytokines, adhesion molecules and nitric oxide; IL-8 is secreted by monocytes and vascular endothelial cells, and its level rapidly increases when inflammation occurs. The relative expression of TNF- α , IL-1 α , IL-6 and IL-8 genes in zebrafish can be detected to evaluate whether the sample has soothing effect.

[0049] Randomly selected zebrafish in 6-well plates, 30 per well. Water-soluble administration of SLS to establish zebrafish skin inflammation model. Water-soluble administration of 0.05% by weight of the calyx saussurea gudzevichii callus extract sample, while setting up a blank group, a model control group, the volume of each well is 3 mL. Three biological repeats. Incubate at 28°C for 18 h in the dark. Extract total RNA from zebrafish in each experimental group, synthesize cDNA, and detect gene expression of β-actin and target genes by q-PCR. Use β-actin as the internal reference for gene expression, and calculate the RNA relative expression of the target gene. The blank group is the gene expression before treatment, and the model group is the gene expression after adding the calyx saussurea gudzevichii callus extract.

[0050] The detection results are shown in Table 6: Table 6. Effect of calyx saussurea gudzevichii callus extract on the relative expression of TNF- α , IL-1 α , IL-6 and IL-8 genes Project name Detection concentration Blank control group Model control group (SLS stimulation) Bract saussurea callus extract TNF- 0.05% 0.211 1 0.305 IL-1 0.05% 0.227 1 0.458 IL-6 0.05% 0.258 1 0.207 IL-8 0.05% 0.239 1 0.667 Compared with the model group, the calyx saussurea gudzevichii callus extract has a significant down-regulation effect on the relative expression of TNF- α , IL-1 α , IL-6 and IL-8 genes, indicating that it has certain anti-inflammatory soothing effect.

[0051] (d) The effect of calyx saussurea gudzevichii callus extract on DNA damage repair Comet assay, also known as single cell gel electrophoresis, is a technology for detecting DNA damage and judging genotoxicity by detecting DNA chain damage, which was first proposed by Ostling in 1984. It can effectively detect and quantitatively analyze the degree of single and double strand breakage of DNA in cells. When various endogenous and exogenous DNA damage factors induce DNA chain breakage, the supercoiled structure is destroyed, and the membrane structure such as cell membrane and nuclear membrane is destroyed under the action of cell lysis solution. Proteins, RNA and other components in the cell diffuse into the cell lysis solution, while the nuclear DNA can only remain in place due to its large molecular weight. Under neutral conditions, DNA fragments can enter the gel and migrate, and under the action of alkaline electrolyte, DNA is denatured and the damaged DNA chain and fragments are released. Because these DNA has small molecular weight and is single-stranded after alkaline denaturation, the negatively charged DNA will migrate from the nuclear DNA to the positive electrode to form a "comet" image, while the undamaged DNA part remains spherical. The more severe the DNA damage, the more chain breaks and fragments are produced, and the smaller the length is. Under the same electrophoresis conditions, the more DNA migrates, the longer the migration distance is. Hydrogen peroxide can cause oxidative damage to DNA, break DNA chain and cause base damage, so by measuring the length of the migrated DNA, the degree of DNA damage in a single cell can be determined, and the protective effect of the sample on DNA damage can be evaluated.

[0052] Randomly selected zebrafish in 6-well plates, 30 per well. Hydrogen peroxide H2O2 was given to establish a zebrafish DNA damage model. The weight ratio of the bract saussurea pinnatifida callus extract sample was 0.01%, and the normal control group and the model control group were set up, and the volume of each well was 3 mL. Replace the liquid once a day, and incubate in the dark at 28°C for 120 h. Collect zebrafish samples, grind and add trypsin for digestion, and perform electrophoresis and staining with a comet assay kit. After staining, 10 zebrafish cells were randomly selected from each experimental group and placed under a fluorescence microscope for photography. The comet analysis software was used to analyze and collect data, and the DNA olive tail moment (tail distance) value (D) of zebrafish cells was analyzed. According to the formula, it is calculated and judged whether the sample has DNA damage repair effect, and the calculation formula is as follows:

[0053] As shown in Table 7, compared with the model control group, the DNA olive tail moment value of the bract saussurea pinnatifida callus extract was significantly reduced, and the DNA damage effect could reach 95% (P<0.01), indicating that it had a certain repair effect on DNA damage. p <0.01), indicating that it had a certain repair effect on DNA damage.

[0054] Table 7. DNA damage repair effect of bract saussurea pinnatifida callus extract Project name Detection concentration DNA tail distance value (D) DNA damage repair effect (%) Normal control group - 1.84 Model control group (H202 stimulation) - 30.8 Bract saussurea callus extract 0.01% 3.20 95 As shown in Table 7, compared with the model control group, the DNA olive tailmoment value of the extract from the callus of Saussurea involucrata was significantly reduced, and its DNA damage repair effect reached 95%. p The value <0.01 indicates that it has a certain repair effect on DNA damage.

[0055] In summary, under different models and with different stimuli, the extract of Saussurea involucrata callus tissue showed superior repair, soothing and anti-inflammatory effects.

[0056] 3. Anti-aging effects of extracts from the callus tissue of Saussurea involucrata bracteata. (a) Effect of Saussurea involucrata callus extract on human dermal fibroblast proliferation Human fibroblasts are the most important cells in the dermal reticular layer of the skin, and are one of the main repair cells after skin aging and cell damage. They not only promote the migration, proliferation and differentiation of epidermal cells, but also secrete a large amount of collagen, elastin and various cell repair factors, and have a strong self-renewal ability, thereby repairing aging skin.

[0057] A sample containing 0.01% by weight of *Saussurea involucrata* callus extract was prepared and added to human fibroblast (FB) culture medium at a volume ratio, with a solvent without the sample serving as a blank control. After 48 h of culture, cells were stained using the CCK8 assay, and absorbance at 450 nm was measured using a microplate reader. The proliferation rate of the blank control was 100%. The effect on FB cell proliferation was calculated to evaluate the anti-aging effect of the sample. The test results are shown in Table 8. Table 8. Effects of Saussurea involucrata callus extract on dermal fibroblast proliferation Project name Detection concentration FB cell viability (%) Blank control - 100% Bract saussurea callus extract 0.01% 111% Bract saussurea plant extract 0.01% 96% As shown in Table 8, at a concentration of 0.01%, the extract of Saussurea involucrata bracteata did not promote the proliferation of dermal fibroblasts, while the extract of Saussurea involucrata bracteata callus tissue significantly promoted the proliferation of human dermal fibroblasts, indicating that it can promote the self-renewal of dermal FB cells and thus has a certain anti-aging effect.

[0058] (b) The inhibitory effect of the extract of Saussurea involucrata callus on β-galactosidase activity β-galactosidase (SA-β-gal) is a marker of cellular senescence; its activity and expression increase in senescent cells. Therefore, the anti-aging effect of a sample can be evaluated by detecting the average staining intensity of β-galactosidase.

[0059] Randomly select zebrafish in 6-well plates, 15 tails per well. Hydrogen peroxide H2O2 is given in water to establish a zebrafish aging model. The weight ratio of 0.01% bract saussurea pulchra callus extract sample is given in water, and the normal control group and model control group are set up, and the volume of each well is 3 mL. Replace the liquid once a day. Incubate at 28°C in the dark for 120 h. Fix the zebrafish with 4% cell tissue fixative overnight, and then use the β-galactosidase staining kit for staining. After staining overnight, randomly select 10 zebrafish from each experimental group under a dissecting microscope, take photos, and use advanced image processing software to analyze and collect data. Analyze the β-galactosidase staining intensity value of zebrafish. The larger the value, the stronger the β-galactosidase activity. The test results are shown in Table 9.

[0060] Table 9. Effect of bract saussurea pulchra callus extract on β-galactosidase activity Project name Detection concentration β-galactosidase staining intensity value (S) β-galactosidase inhibition rate (%) Normal control group - 0.195 - Model control group (H202 stimulation) - 0.261 - Bract saussurea callus extract 0.01% 0.223 58 As can be seen from Table 9, compared with the model control group, the average value of β-galactosidase staining intensity of the bract saussurea pulchra callus extract sample with an addition amount of 0.01% is significantly reduced by 58% (P<0.001), which reveals that the sample has an anti-aging effect. p <0.001), which reveals that the sample has an anti-aging effect.

[0061] (c) Effect of bract saussurea pulchra callus extract on telomerase activity Telomeres have important biological functions. With age, telomere DNA naturally shortens during cell division. When the telomere length becomes very short, the telomere function is impaired, leading to genomic instability, cell aging and apoptosis. Telomeres in somatic cells will gradually shorten with age, at which time telomerase will play a role in delaying telomere shortening, and the maintenance of telomere length requires the presence of telomerase activity. Therefore, aging is closely related to telomerase activity. By detecting telomerase activity, it can be evaluated whether the sample has an anti-aging effect.

[0062] Randomly select zebrafish in 6-well plates, 30 tails per well. Hydrogen peroxide H2O2 is given in water to establish a zebrafish aging model. The weight ratio of 0.01% bract saussurea pulchra callus extract sample is given in water, and the normal control group and model control group are set up, and the volume of each well is 3 mL. Three biological repeats. Replace the liquid once a day. Incubate at 28°C in the dark for 120 h. Collect the sample, grind the homogenate, centrifuge to obtain the supernatant, and use the telomerase activity ELISA kit to determine the OD 450 value of the expression level of telomerase activity in each experimental group. The OD 450 value is used to calculate the telomerase activity.

[0063] Table 10. Effect of bract saussurea pulchra callus extract on telomerase activity Project name Detection concentration Telomerase activity (IU / gprot) Normal control group - 9.10 Model control group (H202 stimulation) - 6.44 Bract saussurea callus extract 0.01% 8.63 The observation found that the telomerase activity of the sample of the calyx leaf of Saussurea involucrata hemi callus extract was increased by 82% compared with the model control group (P < 0.01). p <0.01), which can effectively delay telomere shortening, revealing that the sample of the calyx leaf of Saussurea involucrata hemi callus extract with a weight ratio of 0.01% has an anti-aging effect.

[0064] (d) Effect of calyx leaf of Saussurea involucrata hemi callus extract on the antioxidant enzyme system The aging of zebrafish has some similarities with the aging process of human. Hydrogen peroxide induces DNA damage response through oxidative stress and activates the p53 / p21 signaling pathway, thereby leading to zebrafish aging. When oxidative stress occurs, the ROS in the body of zebrafish increases, the activity of superoxide dismutase (SOD) decreases, and excessive lipid peroxidation product malondialdehyde (MDA) is produced. Catalase (CAT) and glutathione peroxidase (GSH-Px) are important peroxide decomposing enzymes in the body. Glutathione peroxidase (GSH-Px) catalyzes the oxidation of reduced glutathione (GSH) to generate oxidized glutathione (GSSG), thereby protecting the integrity of the cell membrane and protecting cells from oxidative damage. Therefore, the anti-aging effect of the sample can be evaluated by detecting the activity of SOD and the content of GSH.

[0065] Randomly select zebrafish in a 6-well plate, 30 tails per well. Hydrogen peroxide H2O2 is dissolved in water to establish a zebrafish aging model. The sample of calyx leaf of Saussurea involucrata hemi callus extract with a weight ratio of 0.00125% is dissolved in water, and a normal control group and a model control group are set up, with a volume of 3 mL per well. Three biological repeats. Replace the liquid once a day. Incubate in the dark at 28°C for 120 h. After the experiment is completed, collect the zebrafish samples of each group, grind the homogenate, centrifuge to take the supernatant, use the SOD activity detection kit and the GSH content detection kit, use the enzyme label instrument to collect data, and analyze the SOD activity and GSH content in the body of zebrafish. The higher the SOD activity and GSH content, the better the anti-aging effect.

[0066] The detection results are shown in Table 11. Table 11. Effect of calyx leaf of Saussurea involucrata hemi callus extract on the antioxidant enzyme system Project name Detection concentration SOD relative activity (U / mgprot) GSH content (μmol / gprot) Normal control group - 13.0 34.4 Model control group (H202 stimulation) - 7.66 23.1 Bract saussurea callus extract 0.00125% 8.71 26.7 The results in Table 11 show that compared with the model control group, the sample of calyx leaf of Saussurea involucrata hemi callus extract increases the SOD activity and GSH content by 20% (P < 0.05) and 32% (P < 0.01) respectively, revealing that the sample can protect cells from oxidative damage and has an anti-aging effect. p <0.05) and 32% (P < 0.01) respectively, revealing that the sample can protect cells from oxidative damage and has an anti-aging effect. p <0.01), which can effectively delay telomere shortening, revealing that the sample of the calyx leaf of Saussurea involucrata hemi callus extract with a weight ratio of 0.01% has an anti-aging effect.

[0067] In summary, by detecting the effects of the extract of the callus of Saussurea involucrata on the proliferation rate of dermal cells, β-galactosidase activity, telomerase activity, SOD activity and GSH content, it can be shown that the extract of the callus of Saussurea involucrata has anti-aging effect.

[0068] 4 Whitening effect of the extract of the callus of Saussurea involucrata According to the Zebrafish Embryo Method for Determining the Whitening Effect of Cosmetics (T / HPCIA 005-2021), zebrafish were used to evaluate the whitening effect of the callus of Saussurea involucrata. Zebrafish are transparent throughout the early stages of development, and melanin begins to grow from the retinal epithelium when the embryo develops to 24 h. Melanocytes originate from a group of cells differentiated from the dorsal ectoderm - neural crest cells, and then proliferate, migrate and differentiate into pigment cells. Intervention during the process of melanin formation can inhibit the formation of melanin.

[0069] (a) Effect of the extract of the callus of Saussurea involucrata on tyrosinase activity Tyrosinase is a key enzyme in the process of melanin formation. Melanocyte cells located in the basal layer of the epidermis are activated and secrete melanin pigment. Factors such as ultraviolet light stimulate melanin pigment, activate the activity of tyrosinase, and tyrosine is converted to dopa under the action of tyrosinase. Dopa continues to generate dopaquinone under the action of tyrosinase, and dopaquinone is the precursor of brown melanin. At the same time, dopaquinone is converted to dopachrome under the catalysis of tyrosinase, and then most of the dopachrome is converted to dihydroxyindole (DHI) under the action of dopachrome interconvertase, and a small part is converted to dihydroxyindole carboxylic acid (DHICA). Under the action of DHICA oxidase, DHI-melanin and DHICA-melanin, two kinds of true black melanin, are generated. Therefore, the whitening effect of a sample can be evaluated by the activity of tyrosinase.

[0070] Zebrafish tyrosinase activity detection experiment: randomly selected zebrafish in a 6-well plate, 30 per well; water-soluble administration of 0.05% by weight of the extract of the callus of Saussurea involucrata, and a normal control group was set up, with a volume of 3 mL per well. Three biological replicates. Incubate at 28°C in the dark for 45 h. After sampling, use a grinder to homogenize and centrifuge to obtain the supernatant. Use a BCA protein concentration determination kit to determine the protein concentration of each experimental group. Take 250 μg of zebrafish total protein, add 1 mM of levodopa solution at a volume ratio of 1:1, and mix well. Transfer the liquid to a 96-well plate, 200 μL / well, and use a microplate reader to measure the absorbance value at 475 nm wavelength. According to the formula, the whitening effect of the sample is calculated, and the inhibition rate of the sample on zebrafish tyrosinase is the whitening effect, and the calculation formula is as follows:

[0071] The detection results are shown in Table 12: Table 12. Effect of S. involucrata callus extract on tyrosinase activity Project name Detection concentration OD 475 ]] Tyrosinase inhibition rate (%) Significance Normal control group - 0.087 Bract saussurea callus extract 0.05% 0.053 39 <0.001 As shown in Table 12, the S. involucrata callus extract had a tyrosinase activity inhibition rate of 39% at a sample concentration of 0.05% (P < 0.001), indicating that it had a certain whitening effect. p <0.001), indicating that it had a certain whitening effect.

[0072] (b) Effect of S. involucrata callus extract on melanin protein content The zebrafish melanin protein content test was used to evaluate whether the sample had a whitening effect. Zebrafish melanin protein content test: randomly selected zebrafish in a 6-well plate, 30 per well; water-soluble administration of S. involucrata callus extract sample with a weight percentage of 0.05%, and a normal control group was set up, with a volume of 3 mL per well. Three biological replicates; incubate at 28°C in the dark for 45 h. Collect the sample in a 1.5 mL centrifuge tube, add RIPA lysis buffer to each tube, and grind it with a grinder to homogenize, then centrifuge to remove the supernatant. Add 350 μL of 0.2 M NaOH solution to each tube, and incubate at 60°C for 1 h. Prepare a melanin protein standard solution with a certain concentration using 0.2 M NaOH solution. Transfer the standard solution and the sample to be tested to a 96-well plate, 100 μL / well, and use a microplate reader to measure the absorbance value (OD 405 ) at 405 nm, then obtain the melanin protein content (C) of the sample group according to the standard curve, and calculate the inhibition rate of the sample on zebrafish melanin protein according to the formula: the results are shown in Table 13.

[0073] Table 13. Effect of S. involucrata callus extract on melanin protein content Project name Detection concentration Melanin protein content (μg / mL) Melanin protein inhibition rate (%) Significance Normal control group - 37.9 Bract saussurea callus extract 0.05% 21.6 43 <0.01 As shown in Table 13, the S. involucrata callus extract had a melanin protein inhibition rate of 43% at a sample concentration of 0.05% (P < 0.01), indicating that it had a certain whitening effect. p <0.01), indicating that it had a certain whitening effect.

[0074] From the above test results, it can be seen that the S. involucrata callus extract can effectively inhibit the activity of tyrosinase and melanin production in the zebrafish model, indicating that the S. involucrata callus extract with a sample concentration of 0.05% has a certain whitening effect.

[0075] 5 Anti-wrinkle firming effect of S. involucrata callus extract The zebrafish was used to evaluate the firming efficacy of the leafy clematis callus extract according to the team standard “T / ZHCA 015-2022 Evaluation of the firming efficacy of cosmetics by the relative expression of elastin gene of zebrafish larvae”.

[0076] (a) Effect of the leafy clematis callus extract on the relative expression of col1a1a gene The growth, repair, nutrition, elasticity and tension of the skin are related to collagen, and the loss of collagen will reduce the smoothness of the skin and cause wrinkles. In quadrupeds, type I collagen is a trimer, mainly composed of two α1 chains and one α2 chain, which are encoded by col1a1a and col1a2 genes, and performs collagen-related biological functions in connective tissue and bone. There are three type I collagen genes in zebrafish, which encode col1a1a , col1a1b and col1a2 chains of α1 (I), α2 (I) and α3 (I) respectively. Therefore, by detecting the relative expression of col1a1a or (and) col1a1b or (and) col1a2 genes, it can be shown whether the sample has anti-wrinkle efficacy.

[0077] Randomly selected zebrafish in a 6-well plate, 30 per well. Water administration of 0.05% leafy clematis callus extract by weight, while setting up a normal control group, 3 mL per well. Three biological replicates. Incubate at 28°C in the dark for 24 h. Extract total RNA from zebrafish in each experimental group, synthesize cDNA, and detect gene expression of β-actin and target genes by q-PCR. Use β-actin as the internal reference for gene expression, and calculate the RNA relative expression of the target gene. The detection results are shown in Table 14: Table 14. Effect of the leafy clematis callus extract on the relative expression of col1a1a gene Project name Detection concentration Gene relative expression Normal control group - 1.00 Bract saussurea callus extract 0.05% 2.00 The results in Table 14 show that compared with the normal control group, the leafy clematis callus extract at a concentration of 0.05% can increase the relative expression of col1a1a gene to 2 times ( p <0.001), which reveals that it can promote the synthesis of type I collagen. (b) Effect of the leafy clematis callus extract on the relative expression of eln1 gene Elastin is the main component of elastic fibers in skin tissue, which can provide structural support to the skin and prevent the skin from aging and relaxation. Elastin is composed of two types of short peptide segments arranged alternately, eln1 ,eln2 The two genes are responsible for encoding different peptide segments of elastin, and together they regulate the expression level of elastin, making the skin firm and elastic. Zebrafish has similar elastin (elni) genes as humans. Therefore, by detecting the relative expression of elni gene, it can be determined whether the sample has firming efficacy. eln1, eln2 eln1 or (and) eln2 gene. Thus, by detecting the relative expression of

[0078] Randomly select zebrafish in 6-well plates, 30 per well. Water-soluble samples are given, and a normal control group is set up, with a volume of 3 mL per well. Three biological replicates. Incubate at 28°C in the dark for 24 h. Extract total RNA from zebrafish in each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and the target gene. Use β-actin as the internal reference for gene expression to calculate the relative expression of the target gene. The detection results are shown in Table 15: Table 15. Effect of Bellidonna Lactucarium callus extract on the relative expression of eln1 gene Project name Detection concentration Gene relative expression Normal control group - 1.00 Bract saussurea callus extract 0.05% 1.63 The results in Table 15 show that, compared with the normal control group, Bellidonna Lactucarium callus extract at a concentration of 0.05% can increase the relative expression of eln1 gene to 1.63 times ( p <0.05), indicating that it can promote the synthesis of elastin.

[0079] From the above test results, Bellidonna Lactucarium callus extract significantly up-regulates the expression of col1a1a and eln1 genes, promoting the synthesis of type I collagen and elastin in zebrafish, indicating that Bellidonna Lactucarium callus extract has certain anti-wrinkle firming efficacy.

[0080] 6. Acne-removing efficacy of Bellidonna Lactucarium callus extract Acne is a series of inflammatory reactions caused by the skin's inability to properly excrete oil, a chronic inflammatory skin condition that occurs in the hair follicle sebaceous gland tissue, and mainly occurs in the face and chest and back regions where sebum secretion is vigorous. The clinical manifestations of acne vary, including mild comedones, papules, nodules, cysts, and even scars. Propionibacterium acnes is closely related to the occurrence of acne, and it plays a specific role in promoting the release of inflammatory mediators. Propionibacterium acnes induces an inflammatory response in zebrafish, and neutrophils undergo immune response, migrate and accumulate in the epidermis of the skin. The neutrophils of the transgenic neutrophil green fluorescent strain zebrafish (MPX) can be clearly observed under a fluorescence microscope, so the number of neutrophils in the skin of zebrafish can be observed in real time to evaluate whether the sample has acne-removing efficacy. ​

[0081] Zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. A zebrafish inflammation model was established by injection of *Propionibacterium acnes*. A water-soluble extract of *Saussurea involucrata* callus tissue (0.05% by weight) was administered. A normal control group and a model control group were also included, with a volume of 3 mL per well. The plates were incubated at 28°C in the dark for 3 h. Ten zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using advanced image processing software.

[0082] The number of neutrophils (N) in zebrafish was analyzed, and the formula was used to calculate and determine whether the sample had acne-reducing effects. The test results are shown in Table 16. Table 16. Acne-fighting efficacy of Saussurea involucrata callus extract Project name Detection concentration Neutrophil number (N) Anti-acne efficacy % Normal control group - 9.20 - Model control group (Propionibacterium acnes stimulation) - 26.9 - Bract saussurea callus extract 0.05% 19.4 42 The results in Table 16 show that, compared with the model control group, the extract of Saussurea involucrata callus tissue at a weight percentage of 0.05% significantly reduced the number of neutrophils and effectively inhibited the inflammatory response induced by Propionibacterium acnes, revealing the potential of this sample to treat acne.

[0083] Antioxidant activity of extract from the callus tissue of Saussurea involucrata bracteata. CellROX® is a DNA dye with weak intrinsic fluorescence. It is oxidized by reactive oxygen species (ROS) within cells, and the oxidation products bind to DNA, producing bright green fluorescence. CellROX® is soluble in organic solvents (such as dimethyl sulfoxide), and since the main component of the zebrafish yolk sac is fat, CellROX® has high permeability in the yolk sac, resulting in significant staining in this area. Menaquinone can generate reactive oxygen species (ROS). When the amount of ROS produced exceeds the body's scavenging capacity, oxidative stress occurs. The fluorescence intensity of the zebrafish yolk sac is used to evaluate whether a sample has antioxidant properties.

[0084] Zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. A zebrafish oxidative stress model was established by water-soluble administration of menaquinone. A water-soluble extract of *Saussurea involucrata* callus tissue (0.00125% by weight) was administered, along with a normal control group and a model control group, with a volume of 3 mL per well. The plates were incubated at 28°C in the dark for 22 h. Zebrafish were stained with a specific ROS fluorescent reagent. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, analyzing the fluorescence intensity (S) of the zebrafish yolk sac. The antioxidant effect of the sample was calculated and determined according to the formula. The results are shown in Table 17.

[0085] Table 17. Antioxidant effect of Bellidonnae Herba callus extract Project name Detection concentration Yolk sac fluorescence intensity (S) Antioxidant effect % Normal control group - 350264 - Model control group (methyl naphthoquinone stimulation) - 860654 - Bract saussurea callus extract 0.00125% 790615 14 As shown in Table 17, compared with the model control group, the Bellidonnae Herba callus extract sample with a weight percentage of 0.00125% can significantly reduce the fluorescence intensity of zebrafish yolk sac, revealing that the sample has an antioxidant effect.

[0086] 8 Hair loss prevention and scalp care effect of Bellidonnae Herba callus extract Skin microcirculation refers to the blood circulation between arterioles and venules. Blood output by the heart first enters each arterial blood vessel, and then enters the capillary blood vessels for blood circulation and material exchange. The blood circulation of the head belongs to the peripheral circulation, i.e., microcirculation. Microcirculation is the base of human body growth and development, and plays a role in regulating metabolism and balancing cell nutrition. When the function of microcirculation is impaired, the supply of nutrients and the discharge of waste will be affected, and for the hair, white hair or hair loss and other head problems may occur. Therefore, stimulating blood circulation in the head is crucial to ensure the supply of nutrients to the head and activate the microcirculation of the head. It is necessary to awaken the capillaries inside the scalp, promote blood flow, improve microcirculation, and ensure that nutrients can reach the scalp with blood. Therefore, the blood flow speed of the test zebrafish can be detected by using a blood flow analyzer to analyze the improvement of microcirculation, so as to evaluate whether the sample has a hair loss prevention and scalp care effect.

[0087] Randomly select zebrafish in a 6-well plate, 15 per well. Water-soluble administration of Bellidonnae Herba callus extract sample with a weight percentage of 0.05%, and set up a normal control group, 3 mL per well. Incubate at 28°C in the dark for 24 h. Randomly select 10 zebrafish from each experimental group and place them in a blood flow heartbeat analyzer to record videos and collect data, analyze the intersegmental blood vessel blood flow speed (V) of zebrafish, and calculate and judge whether the sample has a microcirculation improvement effect according to the formula. The test results are shown in Table 18:

[0088] Table 18. Microcirculation improvement effect of Bellidonnae Herba callus extract Project name Detection concentration Blood flow velocity (V) Improving microcirculation (%) Normal control group - 365 - S. bracteatum callus extract 0.05% 479.48 31 As shown in Table 18, compared with the normal control group, the intersegmental blood vessel blood flow speed of the Bellidonnae Herba callus extract sample with a weight percentage of 0.05% is significantly increased, revealing that the sample has a microcirculation improvement effect.

[0089] 9 Sleep improvement effect of Bellidonnae Herba callus extract (a) Sleep improvement effect of Bellidonnae Herba callus extract on zebrafish gabra1Effect of saussurea involucrata callus extract on relative expression of genes Pentylenetetrazol (PTZ) is an inhibitor of gamma-aminobutyric acid (GABA) and its receptor complex. GABA is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the central nervous system of mammals. It can inhibit the excitability of brain neurons and thus maintain the stability of the brain. A small dose of PTZ can inhibit the synthesis and release of GABA, resulting in brain neurons not being inhibited and becoming excited, thus causing insomnia. When in a state of insomnia, the body's GABA content decreases, and the expression of GABA receptor genes also decreases. gabra1 Therefore, the relative expression of genes in zebrafish can be used to evaluate whether a sample has a sleep-improving effect. gabra1

[0090] Zebrafish were randomly selected in a 6-well plate, 30 per well. The saussurea involucrata callus extract sample with a weight percentage of 0.025% was administered in water, and a normal control group and a model control group were set up, with a volume of 3 mL per well. Three biological replicates were performed. Incubate at 28°C in the dark for 24 h. Except for the normal control group, the rest of the experimental groups were administered PTZ to establish a zebrafish insomnia model. After 1 h of incubation at 28°C, the zebrafish samples were collected, and the total RNA of each experimental group was extracted, cDNA was synthesized, and the gene expression of the target genes was detected by q-PCR. β-actin The RNA relative expression of the target genes was calculated using β-actin as the internal reference for gene expression. The normal control group was the gene expression before treatment, and the model group was the gene expression after stimulation without the addition of saussurea involucrata extract. The detection results are shown in Table 19: Table 19. Effect of saussurea involucrata callus extract on relative expression of genes gabra1 Item name Detection concentration Gene relative expression amount Significance Normal control group - 1.75 Model control group (PTZ stimulation) - 1 S. bracteatum callus extract 0.025% 1.71 <0.001 As shown in Table 19, compared with the model group, the saussurea involucrata callus extract had an up-regulating effect on the relative expression of gabra1 genes, indicating that it had a certain sleep-improving effect.

[0091] (b) Effect of saussurea involucrata callus extract on relative expression of genes mtnr1aa ​​​A small dose of pentetrazole (PTZ) can inhibit the synthesis and release of gamma-aminobutyric acid, causing the brain neurons to lose inhibition and become excited, thus causing insomnia. Melatonin is one of the hormones secreted by the pineal gland in the brain, which can induce natural sleep in humans, and its secretion has a clear circadian rhythm. The pineal gland synthesizes melatonin at night, and the activity of the enzyme increases, and the secretion level of melatonin increases. When melatonin binds to specific receptors, it initiates cell signal transduction, shortens sleep latency and prolongs sleep duration, thereby overcoming sleep disorders, improving biological rhythm disorders and improving sleep quality. When a person is in a state of insomnia, the secretion of melatonin in the body decreases, and the expression of melatonin receptor gene (MTNR1B) mtnr1aa ) decreases. Therefore, the relative expression of zebrafish mtnr1aa gene can be used to evaluate whether the sample has the effect of improving sleep.

[0092] Randomly select zebrafish in a 6-well plate, 30 per well. Water-soluble administration of 0.025% of the weight percentage of the calyx saussurea callus extract sample, and set up a normal control group and a model control group, each with a capacity of 3 mL. Three biological repeats. Incubate at 28°C in the dark for 24 h. Except for the normal control group, the rest of the experimental groups are water-soluble PTZ to establish a zebrafish insomnia model. After 1 h of incubation at 28°C, collect the zebrafish samples, extract the total RNA of the zebrafish in each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and the target gene. Use β-actin as the internal reference for gene expression, calculate the RNA relative expression of the target gene. The normal control group is the gene expression before treatment, and the model group is the gene expression after adding the calyx saussurea extract. The detection results are shown in Table 20: Table 20. Effect of calyx saussurea callus extract on the relative expression of mtnr1aa gene content Item Detection concentration Gene relative expression amount Significance Normal control group - 3.94 Model control group (PTZ stimulation) - 1.00 S. bracteatum callus extract 0.025% 1.65 <0.05 When a person is in a state of insomnia or sleep deprivation, the skin is prone to water loss, and the sebaceous glands secrete profusely, causing oiliness, etc., which can cause the skin to form wrinkles, dullness and pigmentation. Sleep is a necessary condition to ensure good skin condition. As can be seen from the results in Table 20, the calyx saussurea callus extract with a weight percentage of 0.025% can promote the expression of melatonin receptor gene (MTNR1B) mtnr1aa ) and gabra1 gene, which reflects its effect of improving sleep, and has potential application value in skincare products and sleep-aiding health products.

[0093] 10 Enhanced immune effect of calyx saussurea callus extract Zebrafish possess a highly conserved immune system, with immune cell types and morphology similar to humans, including neutrophils, macrophages, and lymphocytes (B cells and T cells). Like mammals, zebrafish have both an innate and adaptive immune system. The innate immune system fully matures and becomes functional 48 hours after fertilization (48 hpf). High doses of vinorelbine tartrate can inhibit bone marrow formation, leading to a decrease in platelets, erythrocytes, neutrophils, and macrophages, ultimately resulting in weakened immunity. Therefore, detecting the fluorescence intensity of macrophages in the zebrafish tail vein can evaluate whether a sample has an immune-enhancing effect.

[0094] Zebrafish of uniform development were selected and injected with vinorelbine, then placed in 6-well plates (15 fish per well). A water-soluble extract of *Saussurea involucrata* callus tissue (0.05% by weight) was administered. A normal control group and a model control group were also included, with 3 mL per well. The plates were incubated at 28°C in the dark for 48 h. Ten zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data. The fluorescence intensity (S) of macrophages in the zebrafish tail vein was analyzed, and the immune-enhancing effect of the sample was calculated and determined according to the following formula:

[0095] Table 21. Effects of Saussurea involucrata callus extract on immune-enhancing effects Item name Detection concentration Tail vein macrophage fluorescence intensity (S) Enhancing immune function (%) Significance Normal control group - 126151 Model control group (vinorelbine stimulation) - 88931 S. bracteatum callus extract 0.05% 106419 47 <0.05 As shown in Table 21, compared with the model control group, the fluorescence intensity of macrophages in the zebrafish tail vein was increased by 47% in the group treated with Saussurea involucrata callus extract. p The value <0.05 indicates that it enhances the cell vitality of macrophages and has the potential to enhance immune function.

[0096] Autophagy efficacy of 11-spared Saussurea involucrata callus extract Autophagy is a ubiquitous self-protective mechanism in eukaryotic cells. Cells contain aging and damaged organelles, incorrectly synthesized proteins, invading bacteria, and macromolecules. Autophagy forms autophagosomes to encapsulate these harmful substances and combines with lysosomes to form autolysosomes, thus breaking down and clearing them, maintaining cellular homeostasis and the recycling of intracellular substances. The protein LC3 plays a crucial role in autophagy. LC3 exists in two forms: during autophagy, the cytoplasmic form (LC3-I) undergoes enzymatic cleavage of a small polypeptide, transforming into the membrane form (LC3-II). Therefore, the increase in the LC3-II / β-actin ratio can be detected by Western blotting to determine whether autophagy levels have increased.

[0097] Randomly select 4-day post-fertilization zebrafish in a 6-well plate, 30 per well. Water-soluble administration of 0.01% by weight of the bud leaf saussurea involucrata callus extract sample, while setting up a normal control group, 3 mL per well. Three biological repeats. Incubate at 28°C for 24 h in the dark. After incubation, lyse the cells using RIPA lysis buffer and extract the protein. Detect the expression amounts of β-actin and LC3-Ⅱ protein by Western Blot method, and the ratio of LC3-Ⅱ / β-acti is the relative expression amount of LC3-Ⅱ protein. The higher the relative expression amount of LC3-Ⅱ protein, the better the autophagy.

[0098] Table 22. Effect of bud leaf saussurea involucrata callus extract on LC3-Ⅱ protein expression Item Detection concentration LC3-Ⅱ protein relative expression amount Normal control group - 0.543 S. bracteatum callus extract 0.01% 0.692 As shown in Table 22, compared with the normal control group, the relative expression amount of LC3-Ⅱ protein increased by 27% (P<0.05), indicating that the bud leaf saussurea involucrata callus extract can improve the autophagy capacity of cells. p <0.05), indicating that the bud leaf saussurea involucrata callus extract can improve the autophagy capacity of cells.

[0099] In summary, the bud leaf saussurea involucrata callus extract prepared by the present application contains rich active ingredients such as flavonoids, polyphenols and proteins, and the content is more than that of plant medicinal material extract; the bud leaf saussurea involucrata callus extract has better moisturizing, soothing and repairing effects, and also has multiple effects such as anti-aging, whitening, anti-wrinkle tightening, acne removal, antioxidant, hair loss prevention, sleep improvement, immune enhancement, and autophagy capacity improvement; the bud leaf saussurea involucrata callus extract of the present application has the advantages of green environmental protection, stable quality and excellent effect, and solves the problems of wild plant resources and difficult development and utilization of bud leaf saussurea.

[0100] The above is a preferred embodiment of the present application, which is used to explain the technical concept and characteristics of the present application, and its purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and it cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A method of preparing a Saussurea involucrata callus extract, the method comprising the steps of, wherein, The method comprises the following steps: a) culture of callus cells: Take the tender buds of Saussurea involucrata, inoculate into the induction medium MS + 6-BA 0.9~1.1 mg / L + NAA 1.9~2.1 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum to induce, and obtain callus; In the proliferation medium MS + 6-BA 1.3~1.7mg / L + NAA 1.8~2.2mg / L + KT1.8~2.2 mg / L + 30g / L sucrose + 3.5 g / L KONJAC, inoculate 1% ~ 2% of the callus for the multiplication culture, and obtain the cell of the bract saussurea obvallata callus; b) preparation of Saussurea involucrata callus extract: Add the callus cells obtained after value-added cultivation of Saussurea involucrata into water for extraction to obtain the Saussurea involucrata callus extract.

2. The production method according to claim 1, wherein The induction medium is: MS + 6-BA 1.0 mg / L + NAA 2.0 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum, the proliferation medium is MS + 6-BA 1.5 mg / L + NAA 2.0 mg / L + KT 2.0 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum, and the callus culture conditions are: temperature 20±1℃, humidity 50%~70%, and dark culture.

3. The production method according to claim 1, wherein The preparation of the Saussurea involucrata callus extract comprises the following steps: A, weigh the callus cells of Saussurea involucrata, grind and crush; B. Add 10 mL of cell suspension of B. subtilis to 10 mL of cell suspension of E. coli ~ 30 volumes of deionized water, preferably 20 volumes of deionized water, submerged stirring; C, 550 ~ 650 W, preferably 600 W ultrasonic extraction, centrifugal filtration, taking the supernatant, and combining the filtrates; D, dry to obtain the Saussurea involucrata callus extract.

4. The extract of Saussurea involucrata callus obtained by the preparation method according to any one of claims 1 to 3, characterized in that, The total polyphenol content in the callus of Saussurea involucrata is 35~45 mg / g dry weight, the flavonoid content is 55~65 mg / g dry weight, and the protein content is 535~545 mg / g dry weight.

5. The Saussurea involucrata callus extract of claim 4, wherein, The total polyphenol content in the callus of Saussurea involucrata is 40.34 mg / g dry weight, the flavonoid content is 59.42 mg / g dry weight, and the protein content is 541.93 mg / g dry weight.

6. Use of the Saussurea involucrata callus extract of claim 4 in the preparation of skin external agents, hair external agents, beauty oral agents, health products, foods or food additives for moisturizing, soothing and repairing, anti-aging, whitening, tightening and anti-wrinkling, acne removal, antioxidant, hair loss prevention and scalp care, sleep improvement, immunity enhancement, and autophagy capacity improvement.

7. Use according to claim 6, wherein The moisturizing effect is to promote aqp3a gene expression and promote hyaluronic acid synthesis; the soothing and repairing effect is to repair skin UV damage, promote ATP content, repair DNA damage, and inhibit the expression of TNF-α, IL-1α, IL-6 and IL-8 inflammatory factors; the anti-aging effect is to promote the proliferation of human dermal fibroblasts, inhibit β-galactosidase activity, promote telomerase activity, increase superoxide dismutase (SOD) activity, and increase glutathione (GSH) content; the whitening effect is to inhibit the tyrosinase activity and melanin production of human epidermal melanocyte B16; the firming and anti-wrinkle effect is to increase the expression of elastin eln2 gene and col1a1a gene; the acne-removing effect is to reduce neutrophil content; the sleep-improving effect is to promote the expression of melatonin receptor gene (mtnr1aa) and / or GABA receptor gene ( gabra1 The immune-enhancing effect is to increase macrophage activity; the anti-hair loss and scalp care effects are to enhance blood microcirculation; and the autophagy effect is to increase the expression level of LC3-II protein.

8. A skin external preparation, characterized by comprising: In the skin external agent, the content of the Saussurea involucrata callus extract is 0.001%~1%, and the percentage refers to the percentage of the mass of the Saussurea involucrata callus extract in the total mass of the skin external agent; preferably, the content of the Saussurea involucrata callus extract is 0.01%~1%.

9. A cosmetic oral agent, characterized by, In the beauty oral agent, the content of the Saussurea involucrata callus extract is 0.001%~1%, and the percentage refers to the percentage of the mass of the Saussurea involucrata callus extract in the total mass of the beauty oral agent; preferably, the content of the Saussurea involucrata callus extract is 0.01%~1%.

10. A health food, characterized by, In the health product, the content of the Saussurea involucrata callus extract is 0.001%~1%, and the percentage refers to the percentage of the mass of the Saussurea involucrata callus extract in the total mass of the health product; preferably, the content of the Saussurea involucrata callus extract is 0.01%~1%.

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