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

Abstract

The invention relates to an application of a saussurea longifolia callus extract in the aspects of preparing a skin external preparation, a beautifying oral preparation, a health care product, food or a food additive and the like, and the saussurea longifolia callus extract has excellent effects of resisting aging, improving sleep and enhancing immunity. Meanwhile, the composition also has multiple effects of relieving, repairing, whitening, resisting inflammation, tightening, resisting wrinkles, moisturizing, removing acnes, resisting oxidation, preventing hair loss, caring hair, caring scalp and the like, and can be used as an active additive to be applied to skin and hair care products and foods. The saussurea longifolia callus cells are used as the raw material, 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 used for production 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] This invention belongs to the field of daily-use product technology, and in particular relates to an extract of callus tissue from Saussurea longifolia, its preparation method, and its application. Background Technology

[0002] Long-leaved snow lotus ( Saussurealongi folia *Saussurea* Franch. is a plant belonging to the genus *Saussurea* in the family Asteraceae. The plant grows to a height of 15-30 cm, with an erect stem covered in long, white, soft hairs. It grows in alpine meadows and thickets at altitudes of 3400-4500 m. The whole plant can be used medicinally, possessing properties such as clearing heat and detoxifying, reducing swelling and removing blood stasis.

[0003] In addition, there are no research reports on the application of Saussurea longifolia and its callus extracts in skin care, hair care, and health food. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing an extract of Saussurea longifolia callus tissue and its application.

[0005] One aspect of the present invention provides a method for preparing an extract from callus tissue of Saussurea longipes, wherein the method comprises the following steps: a) Culture of callus cells: Young shoots of *Saussurea longifolia* were taken and inoculated onto MS medium with 0.3 g of 2,4-D. ~ 0.7 mg / L + 6-BA 1.8 ~ Callus tissue was obtained by induction with 2.2 mg / L sucrose, 30 g / L sucrose, and 3.5 g / L gellan gum. In proliferation medium B5 / MS / SH+6-BA 0.5 ~ 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or B5 / MS + mT 0.8-1.2 mg / L + 2,4-D 0.3 mg / L ~ Inoculate 1% of the growth medium volume with 0.7 mg / L sucrose, 30 g / L sucrose, and 3.5 g / L gellan gum. ~ 2% of the callus tissue was used for proliferation culture to obtain the callus cells of the long-leaved snow lotus; b) Preparation of extract from callus tissue of Saussurea longifolia: The callus cells of the long-leaved snow lotus were added to water for extraction to obtain the long-leaved snow lotus callus extract.

[0006] Further, the induction medium is: MS + 2,4-D 0.5 mg / L + 6-BA 2.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, and the proliferation medium is...： B5 / MS / SH + 6-BA 0.5-1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or B5 / MS + mT 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L.

[0007] The callus culture conditions are as follows: temperature 20±1℃, humidity 50%~70%, 12 hours of light per day, and light intensity of 1500 Lx ~ 2000 Lx.

[0008] Using these culture conditions and medium, the callus induction rate is high, and the proliferation coefficient is large, without waterlogging or browning. High-quality, large-volume *Saussurea longipes* callus can be obtained.

[0009] Furthermore, the extraction of callus cells from *Saussurea longipes* includes the following steps: A. Weigh the callus cells of the long-leaved snow lotus and grind them into pieces; B. Add 10g of long-leaved snow lotus callus cells. ~ Immerse and stir with 30 times the volume of deionized water, preferably 20 times the volume; C, 550 ~ Ultrasonic extraction at 650W, preferably 600W, followed by centrifugation and filtration. The supernatant was then filtered, and the filtrates were combined. D. Drying to obtain the long-leaved snow lotus callus extract.

[0010] This invention also provides a *Saussurea longifolia* callus extract, wherein the *Saussurea longifolia* callus extract is obtained by the above-described preparation method, and the total polyphenol content in the *Saussurea longifolia* callus is 13%. ~ 23 mg / g, flavonoid content is 16 ~ 28mg / g, protein content is 168 ~ 178mg / g.

[0011] Furthermore, the total polyphenol content of the *Saussurea longifolia* callus was 18.1 mg / g, the flavonoid content was 21.5 mg / g, and the protein content was 173 mg / g.

[0012] The total polyphenol, flavonoid, and protein content in the callus tissue of *Saussurea longifolia* obtained by this invention is significantly higher than that in *Saussurea longifolia* plants, indicating that it is rich in active ingredients.

[0013] This invention also provides the application of long-leaved snow lotus callus extract in the preparation of skin topical agents, hair care topical agents, oral beauty agents, health products, food or food additives with anti-aging, soothing and repairing, whitening, anti-inflammatory, firming and anti-wrinkle, moisturizing, acne removal, sleep improvement, anti-oxidation, immune enhancement, anti-hair loss and hair care and scalp care effects.

[0014] The anti-aging effects include promoting the proliferation of human dermal fibroblasts, inhibiting β-galactosidase activity, promoting telomerase activity, promoting superoxide dismutase (SOD) activity, and increasing glutathione (GSH) content. The repair effects include repairing skin damage from UV exposure and increasing ATP content, thus repairing DNA damage. The soothing effect includes reducing irritation caused by acetic acid. The anti-inflammatory effect includes inhibiting the expression of inflammatory factors such as TNF-α, IL-1α, IL-6, and IL-8. The whitening effect includes inhibiting tyrosinase activity and melanin production in human epidermal melanocytes (B16). The firming and anti-wrinkle effects include increasing the expression of the elastin eln1 gene and the col1a1a gene. The moisturizing effect includes promoting the expression of the aqp3a gene and the synthesis of hyaluronic acid. The acne-reducing effect includes reducing neutrophil content. The sleep-improving effect includes promoting the expression of the melatonin receptor gene mtnr1aa and GABA. The receptor gene gabra1 is expressed; the immune-enhancing effect is to increase macrophage activity; the hair loss prevention and scalp care effects are to enhance blood microcirculation.

[0015] The present invention also provides a topical skin agent, wherein the content of the long-leaved snow lotus callus extract in the topical skin agent is 0.001%. ~ The percentage of 1% refers to the mass of the *Saussurea longifolia* callus extract relative to the total mass of the topical skin agent. Preferably, the content of the *Saussurea longifolia* callus extract is 0.01%. ~ 1%.

[0016] The present invention also provides a cosmetic oral preparation, wherein the content of the long-leaved snow lotus callus extract in the cosmetic oral preparation is 0.001%. ~ The percentage of 1% refers to the mass of the *Saussurea longifolia* callus extract relative to the total mass of the oral cosmetic preparation. Preferably, the content of the *Saussurea longifolia* callus extract is 0.01%. ~ 1%.

[0017] The present invention also provides a health food product, wherein the content of the long-leaved snow lotus callus extract in the health food product is 0.001%. ~The percentage of 1% refers to the percentage of the mass of the *Saussurea longifolia* callus extract to the total mass of the health food product. Preferably, the content of the *Saussurea longifolia* callus extract is 0.01%. ~ 1%.

[0018] Beneficial effects The callus extract of Saussurea longifolia obtained by this invention has superior anti-aging, sleep improvement and immune enhancement effects. It also has multiple effects such as soothing and repairing, anti-photoaging, whitening, moisturizing, firming and anti-wrinkle, acne removal and anti-oxidation. It can be used as an active additive in skin care products.

[0019] The long-leaved snow lotus callus extract prepared by this invention has the advantages of being green and environmentally friendly, having stable quality, and having excellent efficacy, thus solving the problem of the scarcity and difficulty in developing and utilizing wild long-leaved snow lotus resources. Attached Figure Description

[0020] Figure 1 Snow lotus plant, callus tissue and medicinal materials prepared from it Figure 2 Immunostimulation assay of Saussurea longifolia callus extract: Fluorescent staining of macrophages from zebrafish tail veins. Detailed Implementation

[0021] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0022] Experimental methods in the following examples that do not specify specific conditions should be performed according to conventional methods and conditions, or according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0023] Example 1: Culture and Extraction of Callus from Saussurea longipes 1) Culture medium preparation and culture conditions Choose one of MS, B5, White, Heller, or SH medium, and prepare the medium by adding different plant growth regulators and cytokinins according to a certain ratio. Add 30 g / L sucrose and 3.5 g / L gellan gum to each medium, adjust the pH to 5.7-5.8, and sterilize at 121℃ for 20 min in an autoclave. All plant explants were cultured under the following conditions.

[0024] 20±1℃, humidity 50%~70%, 12 hours of light per day, light intensity 1500 Lx ~ 2000 Lx.

[0025] The basal culture media MS (M519), B5 (G398), SH (816) and plant growth regulators were all purchased from Pytotechnolagy.

[0026] Plant growth regulator instructions: NAA: Naphthaleneacetic acid 6-BA: 6-Benzylaminopurine 2,4-D: 2,4-Dichlorophenoxyacetic acid mT: Meta-topolin, cytokinin Explant disinfection and sterile seedling culture

[0027] Take tender shoots and young leaves of wild snow lotus, rinse them under running water for 2 hours, then disinfect them with 75% ethanol for 30 seconds in a sterile laminar flow hood, and rinse them three times with sterile water. Treat them with 10% (v / v) NaClO solution in a shaker at 110 rpm for 10 min, or with 5% (v / v, dissolved in 3XMS liquid medium) PPM (Plant Preservation Mixture) solution, immerse them in a shaker at 110 rpm for 1 h, and finally rinse them three times with sterile water for later use.

[0028] 3) Induction and proliferation of callus tissue

[0029] B5 / MS / SH + 6-BA 0.5 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or B5 / MS + mT 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, cultured for 30 days, the callus proliferation coefficient was above 5, and a large amount of callus tissue was obtained by continuous subculturing.

[0030] 4) Extraction, preparation, and detection of active ingredients (a) Preparation of callus cell extract from Saussurea longifolia Callus tissue and medicinal plant of Saussurea longifolia were taken, crushed and added to 20 times water. The mixture was extracted by ultrasonication (600W) for 30 minutes, filtered and the filtrate was collected to obtain the plant extract of Saussurea longifolia callus tissue and medicinal plant. The contents of polyphenols, flavonoids and proteins were then tested.

[0031] (b) Determination of total polyphenols Standard curve: Gallic acid 0.33 mg / mL solution was successively diluted to concentration gradients of 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, and 0.3300 mg / mL. 100 μL of *Saussurea longifolia* callus cell extract was added to 500 μL of 10% Folin-Ciocalteu reagent, mixed thoroughly, and incubated for 5 min (not exceeding 8 min). Then, 400 μL of 7.5% Na2CO3 was added, mixed thoroughly, and incubated at room temperature for 60 min. Finally, 100 μL was measured at 765 nm using a microplate reader. The total polyphenol content of *Saussurea longifolia* callus was calculated based on the standard curve, and the results are shown in Table 1: the total polyphenol content of *Saussurea longifolia* callus was 18.1 mg / g dry weight, and the total polyphenol content of *Saussurea longifolia* medicinal material was 2.99 mg / g dry weight.

[0032] (c) Determination of total flavonoids Standard solution: A 0.2315 mg / mL ethanol solution was prepared with quercetin and diluted sequentially to a concentration gradient of 2-10 times. Take 300 μL of the extract from the callus cells of *Saussurea longifolia*, add 50 μL of 5% NaNO2, mix, and incubate at room temperature for 6 min; then add 50 μL of 10% Al(NO3)3, mix, and incubate at room temperature for 6 min; finally, add 500 μL of 4% NaOH and 140 μL of water, mix, and incubate at room temperature for 15 min. Take 100 μL of the mixture and measure the OD value at 500 nm. The total polyphenol content of *Saussurea longifolia* was calculated based on the standard curve, and the results are shown in Table 1: the total flavonoid content of the *Saussurea longifolia* callus was 21.5 mg / g dry weight, and the total flavonoid content of the *Saussurea longifolia* medicinal material was 7.30 mg / g dry weight.

[0033] (d) Protein assay Protein content was determined using a BCA protein concentration assay kit: 20 μL of sample was added to 200 μL of BCA working solution, incubated at 37ºC for 20-30 minutes, and the absorbance was measured at 562 nm. The protein content in the sample was calculated based on the BSA standard solution curve. The results are shown in Table 1: the protein content of *Saussurea longifolia* callus was 173 mg / g dry weight, and the protein content of *Saussurea longifolia* medicinal material was 25.6 mg / g dry weight.

[0034] Table 1. Content of active ingredients in Saussurea longipeda minima project Total polyphenol content (mg / g) Total flavonoid content (mg / g) Protein content (mg / g) Callus tissue of Saussurea longifolia 18.1 21.5 173 long-leaved snow lotus medicinal material 2.99 7.30 25.6 In summary, the callus tissue of *Saussurea longifolia* contains abundant polyphenols, flavonoids, and proteins, with higher levels than the medicinal plant itself. Furthermore, LC-MS analysis revealed that the aqueous extract of *Saussurea longifolia* callus tissue contains active ingredients such as syringin, chlorogenic acid, and adenosine.

[0035] Example 2: Efficacy Experiment of Callus Extract from Saussurea longifolia 1. Anti-aging effects of Saussurea longipes callus extract (a) Effect of Saussurea longifolia 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.

[0036] The prepared *Saussurea longifolia* callus extract sample was added to human fibroblast (HDF) culture medium according to the calculated concentration based on the solid content of the extract, with a solvent without the sample serving as a blank control. After 48 hours of culture, cells were stained using the MTT assay, and the absorbance at 550 nm was measured using a microplate reader. The proliferation rate of the blank control was 100%, and the effect on FB cell proliferation was calculated to evaluate the anti-aging effect of the sample. The results of the effect of *Saussurea longifolia* callus extract on dermal fibroblast proliferation are shown in Table 2. Table 2. Effects of Saussurea longifolia callus extract on dermal fibroblast proliferation. Project Name Test concentration (weight percentage) HDF cell viability (%) Blank control - 100 Snow lotus callus extract 0.0015% 123 Snow lotus callus extract 0.015% 146 Snow lotus callus extract 0.02% 140 extract of Saussurea longifolia medicinal material 0.02% 133 Snow lotus callus extract 0.04% 173 extract of Saussurea longifolia medicinal material 0.04% 146 The results showed that the extract of *Saussurea longifolia* callus tissue at concentrations of 0.0015%-0.04% by weight could promote the proliferation of human dermal fibroblasts. Furthermore, at test concentrations of 0.02% and 0.04%, its effect was superior to that of the original herbal extract. This indicates that the extract of *Saussurea longifolia* callus tissue has a superior anti-aging effect, and its effect is better than that of the original herbal extract.

[0037] (b) The inhibitory effect of Saussurea longifolia callus extract 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.

[0038] Zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. A zebrafish aging model was established by water-soluble hydrogen peroxide. Water-soluble samples were administered, along with a blank control group and a model control group, with a volume of 3 mL per well. The liquid was changed daily. The plates were incubated at 28 ℃ in the dark for 120 h. The zebrafish were fixed overnight with 4% cell tissue fixative and stained using a β-galactosidase staining kit. After staining overnight, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data, analyzing the β-galactosidase staining intensity value. A higher value indicates stronger β-galactosidase activity. The results of the test on the effect of *Saussurea involucrata* callus extract on β-galactosidase activity are shown in Table 3. Table 3. Effects of Saussurea longifolia callus extract on β-galactosidase activity Project Name Sample concentration (by weight) Average β-galactosidase staining intensity (pixels) Blank control group - 0.195 Model control group (hydrogen peroxide stimulation) - 0.261 Snow lotus callus extract 0.006% 0.218 As shown in Table 3, the average staining intensity of β-galactosidase in the callus extract of Saussurea longifolia at a concentration of 0.006% by weight was significantly reduced compared with that of the model control group, indicating that the sample can significantly inhibit the activity of β-galactosidase in cells and has certain anti-aging potential.

[0039] c) Effects of Saussurea longifolia callus extract on telomerase activity Telomeres play a vital biological role. As we age, telomere DNA naturally shortens during somatic cell division. When telomere length becomes extremely short, telomere function is impaired, leading to genomic instability, cellular senescence, and apoptosis. Skin telomeres gradually shorten with age, and telomerase plays a crucial role in slowing this shortening. Maintaining telomere length requires the presence of telomerase activity. Therefore, aging is closely related to telomerase activity. Measuring telomerase activity can evaluate whether a sample possesses anti-aging properties.

[0040] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. A zebrafish aging model was established by water-soluble administration of hydrogen peroxide. A water-soluble extract of *Saussurea longifolia* callus tissue was administered at a weight percentage of 0.006%, with a blank control group and a model control group included. Each well contained 3 mL of extract. Three biological replicates were performed. The liquid was changed daily. The plates were incubated at 28 ℃ in the dark for 120 h. Samples were collected, homogenized, and the supernatant was collected by centrifugation. Telomerase activity was measured using a telomerase activity ELISA kit. Data were collected using a microplate reader, and the OD450 values ​​of telomerase activity expression levels in each experimental group were analyzed. Telomerase activity was calculated based on the OD450 values. The results are shown in Table 4.

[0041] Table 4. Effects of Saussurea longifolia callus extract on telomerase activity Project Name Sampling concentration Telomerase activity (IU / gprot) Blank control group - 9.103 Model control group (hydrogen peroxide stimulation) - 6.436 Snow lotus callus extract 0.006% 8.894 The results showed that the telomerase activity of the callus extract sample of Saussurea longifolia at a weight percentage of 0.006% was significantly increased by 38% compared with the model control group, indicating that the sample has a certain anti-aging effect.

[0042] (d) Effect of Saussurea longifolia callus extract on enhancing superoxide dismutase (SOD) activity Hydrogen peroxide induces DNA damage through oxidative stress and activates the p53 / p21 signaling pathway, leading to aging in zebrafish. Oxidative stress increases ROS levels in zebrafish, decreases superoxide dismutase (SOD) activity, and produces excessive malondialdehyde (MDA), a lipid peroxidation product. Catalase (CAT) and glutathione peroxidase (GSH-Px) are important peroxidases in vivo. Glutathione peroxidase (GSH-Px) catalyzes the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG), thereby protecting cell membrane integrity and protecting cells from oxidative damage. Therefore, detecting SOD activity can evaluate the anti-aging effects of samples.

[0043] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. A zebrafish aging model was established by water-soluble administration of hydrogen peroxide. Samples were administered water-solublely, and a blank control group and a model control group were also included, with a volume of 3 mL per well. Three biological replicates were performed. The liquid was changed daily. The plates were incubated at 28 ℃ in the dark for 120 h. After the experiment, zebrafish samples from each group were collected, homogenized, centrifuged, and the supernatant was collected. Data on SOD activity in the zebrafish were acquired using a microplate reader and analyzed using a SOD activity assay kit. The test results are shown in Table 5. Table 5. Effects of Saussurea longifolia callus extract on superoxide dismutase (SOD) activity. Project Name Sample concentration (by weight) SOD activity (U / mgprot) Blank control - 13.040 Model control group (hydrogen peroxide stimulation) - 7.657 Snow lotus callus extract 0.00125% 8.813 The results in Table 5 show that the SOD activity of the extract from the callus tissue of Saussurea longifolia was significantly enhanced compared with that of the model control group at a concentration of 0.00125% by weight, indicating that the sample has a certain anti-aging effect.

[0044] (e) The effect of the long-leaved snow lotus callus extract on promoting the increase of glutathione (GSH) content.

[0045] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. A zebrafish aging model was established by water-soluble hydrogen peroxide. Samples were administered water-solublely, and a blank control group and a model control group were also set up, with a volume of 3 mL per well. Three biological replicates were performed. The liquid was changed daily. The plates were incubated at 28 ℃ in the dark for 120 h. After the experiment, zebrafish samples from each group were collected, homogenized, centrifuged, and the supernatant was collected. Data were collected using a GSH content detection kit and a microplate reader to analyze the GSH content in the zebrafish. Higher GSH content indicated better anti-aging effects. The results are shown in Table 6.

[0046] Table 6. Effects of Saussurea longifolia callus extract on glutathione (GSH) content. Project Name Sample concentration (by weight) Glutathione (GSH) content (μmol / gprot) Blank control group - 34.4 Model control group (hydrogen peroxide stimulation) - 23.1 Snow lotus callus extract 0.00125% 28.3 The results in Table 6 show that the GSH content of the *Saussurea longifolia* callus extract at a concentration of 0.00125% by weight was significantly increased compared with the model control group, indicating that the sample has a certain anti-aging effect.

[0047] The anti-aging test results show that the extract of Saussurea longifolia callus tissue significantly promotes the proliferation of human dermal fibroblasts, enhances the activity of telomerase and superoxide dismutase, increases glutathione content, and inhibits β-galactosidase activity, revealing that the extract of Saussurea longifolia callus tissue has good anti-aging effects.

[0048] 2. The soothing and repairing effects of extracts from the callus tissue of Saussurea longipeda minima. (a) Effects of Saussurea longifolia callus extract on UV-damaged cell repair and ATP synthesis promotion Skin aging is divided into intrinsic aging and extrinsic aging. Sunlight, especially ultraviolet (UV) radiation, is the main factor in the formation of extrinsic aging, hence extrinsic aging is also known as photoaging. UVA (320nm-400nm) and UVB (280-320nm) are associated with skin photoaging. Numerous studies have shown that ultraviolet radiation can induce apoptosis in fibroblasts, altered mitochondrial DNA, and an increase in intracellular reactive oxygen species. A photodamage model of the skin was established by irradiating human immortalized epidermis (Hacat) and human fibroblasts (FB) with UVB. This model was used to evaluate the repair and anti-photoaging effects of the samples.

[0049] Human immortalized epidermal (Hacat) cells and human fibroblasts were seeded at 12,000 cells / well and 6,000 cells / well, respectively, in 96-well plates and cultured for 24 hours in a 5% CO2 cell culture incubator at 37°C. Afterward, the cells were cultured in medium containing the sample for 24 hours, then the medium was replaced with PBS, and the cells were irradiated with UVB at a dose of 40 mJ / cm². 2Then, culture medium containing the test sample was added, and the cells were cultured for another 24 hours. Cell viability was then detected using the CCK8 assay. The untreated group served as the blank control group, and the UVB irradiation group without the sample served as the model control group.

[0050] Human immortalized epidermal (Hacat) cells were seeded at 0.5 M per well in 6-well plates and cultured for 24 hours in a 5% CO2 cell culture incubator at 37°C. Afterward, the cells were cultured in culture medium for 24 hours, then the medium was replaced with PBS, followed by UVB irradiation. Culture medium containing the test sample was then added, and the cells were cultured for another 24 hours. The culture medium was removed, and cell lysis buffer was added for 3-5 minutes. After centrifugation, the supernatant was used for ATP content testing. The UVB irradiation group without the test sample served as a blank control. The results are shown in Tables 7 and 8.

[0051] Table 7. Effects of Saussurea longifolia callus extract on UV radiation damage to skin cells. Project Name Test concentration Hacat cell viability (%) FB cell viability (%) Blank control - 100 100 Model control group (UVB radiation) - 41 72 Snow lotus callus extract 0.01% 47 88 extract of Saussurea longifolia medicinal material 0.01% 33 66 Snow lotus callus extract 0.03% 65 110 extract of Saussurea longifolia medicinal material 0.03% 33 71 Table 8. Effects of Saussurea longifolia callus extract on intracellular ATP synthesis.

[0052] As shown in Tables 7 and 8, the Hacat and FB cell viability of cells treated with *Saussurea longifolia* callus extract were significantly enhanced compared to the model control group. *Saussurea longifolia* callus extract at concentrations of 0.01%–0.05% exhibited a significant protective effect against UVB-induced skin cell damage, with effects significantly superior to *Saussurea longifolia* herbal extract at the same concentration. Furthermore, *Saussurea longifolia* callus extract at concentrations of 0.015%–0.03% by weight promoted ATP synthesis. These results collectively demonstrate the repair and anti-photoaging effects of the samples.

[0053] (b) Effect of Saussurea longifolia callus extract on reduced movement distance in zebrafish Glacial acetic acid has strong penetrating and corrosive properties. Skin contact with glacial acetic acid will cause a stinging and burning sensation. Zebrafish whose swimming ability is not yet fully developed were selected, and glacial acetic acid was used to stimulate the zebrafish to induce inflammatory pain over a large area and for a long time. The more movement traces the zebrafish made, the more severe the pain became. A behavioral analyzer was used to measure the total movement distance of the zebrafish to evaluate whether the sample had a soothing and repairing effect.

[0054] Zebrafish were randomly selected and placed in 6-well plates, one fish per well. The sample was administered in water, and a blank control group and a model control group were also set up, with a volume of 3 mL per well. The plates were incubated at 28℃ in the dark for 24 h. Ten zebrafish from each experimental group were randomly selected and transferred to 96-well plates, where glacial acetic acid was administered in water to establish a zebrafish pain model. The movement trajectory of the zebrafish was immediately measured using a behavior analyzer, and the total movement distance (D) was analyzed and collected. The efficacy of the sample in relieving and repairing pain was calculated and determined according to the formula. The test results are shown in Table 9.

[0055]

[0056] Table 9. Effects of Saussurea longifolia callus extract on zebrafish movement distance. Project Name Sample concentration (by weight) Total distance traveled (D) Soothing effect (%) Blank control group - 115 - Model control group (stimulated by glacial acetic acid) - 5663 - Snow lotus callus extract 0.03% 3884 28% The results in Table 9 show that, compared with the model control group, the total movement distance of zebrafish was significantly reduced at a concentration of 0.03% by weight of Saussurea longifolia callus extract, indicating that Saussurea longifolia callus at this concentration can alleviate the irritation caused by acetic acid and has a soothing effect.

[0057] (c) Inhibitory effect of Saussurea longifolia callus extract on the expression of inflammatory factors Sodium dodecyl sulfate (SLS) may trigger a stimulating response in the body. When this stimulant enters the zebrafish, it induces an inflammatory response, causing neutrophils to initiate an immune response, migrate to the skin epidermis, and aggregate. TNF-α is the earliest and most important inflammatory cytokine to appear during the inflammatory response, activating neutrophils. IL-1 plays a crucial role in transmitting information, activating and regulating immune cells, and promoting the proliferation and differentiation of inflammatory cells. IL-1 exists in two different molecular forms, IL-1α and IL-1β. IL-16 can promote the upregulation of neutrophil function and 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 during inflammation. The soothing efficacy of a sample can be evaluated by detecting the relative expression levels of TNF-α, IL-1α, IL-6, and IL-8 genes in zebrafish.

[0058] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. A zebrafish skin inflammation model was established by water-soluble administration of SLS. Water-soluble samples were administered, and a blank control group and a model control group were also included. The volume of each well was 3 mL.

[0059] Three biological replicates were performed. The cells were incubated at 28 °C in the dark for 18 h. Total RNA was extracted from zebrafish in each experimental group, and cDNA was synthesized. The expression of β-actin and the target gene was detected by q-PCR. β-actin was used as an internal control for gene expression, and the relative RNA expression level of the target gene was calculated. The blank group represents the gene expression level before treatment, and the model control group represents the gene expression level after stimulation without the addition of *Saussurea longifolia* callus extract. The sample groups used samples with 0.03% (w / w) *Saussurea longifolia* callus extract. The results are shown in Table 10.

[0060] Table 10 Effects of Saussurea longifolia callus extract on the relative expression levels of TNF-α, IL-1α, IL-6 and IL-8 genes. Project Name Sample concentration (by weight) TNF-α IL-1α IL-6 IL-8 Blank control group - 0.211 0.227 0.258 0.239 Model control group (stimulated with sodium dodecyl sulfate) - 1.003 1.000 1.000 1.000 Snow lotus callus extract 0.03% 0.248 0.356 0.126 0.366 The results in Table 10 show that, compared with the model control group, the relative expression levels of TNF-α, IL-1α, IL-6 and IL-8 genes were downregulated at a concentration of 0.03% by weight of the Saussurea longifolia callus extract. Therefore, the Saussurea longifolia callus extract at this concentration has a certain inhibitory effect on the inflammatory response induced by sodium dodecyl sulfate (SLS) and has a certain anti-inflammatory and soothing effect.

[0061] (d) Effect of Saussurea longifolia callus extract on DNA damage repair The comet assay, also known as single-cell gel electrophoresis, is a technique first proposed by Ostling in 1984 to determine genotoxicity by detecting DNA strand damage. It effectively detects and quantifies the degree of single- and double-strand breaks in DNA within cells. When various endogenous and exogenous DNA damaging factors induce DNA strand breaks, the supercoiled structure is disrupted. Under the influence of cell lysis buffer, membrane structures such as the cell membrane and nuclear membrane are damaged, and intracellular proteins, RNA, and other components diffuse into the lysis buffer, while nuclear DNA, due to its large molecular weight, remains in situ. Under neutral conditions, DNA fragments can migrate into the gel, while under the influence of alkaline electrolytes, the DNA unwinds, releasing the damaged DNA strands and fragments. Because these DNA molecules are small and denatured into single strands by alkaline methods, negatively charged DNA migrates away from the nuclear DNA towards the positive electrode during electrophoresis, forming a "comet"-like pattern, while the undamaged DNA remains spherical. The more severe the DNA damage, the more strand breaks and fragments are produced, and the shorter the fragments are. Under the same electrophoretic conditions, the more DNA migrates and the longer the migration distance. Hydrogen peroxide can cause oxidative damage to DNA, disrupting DNA strand breaks and base damage. Therefore, by measuring the length of the migrated DNA portion, the degree of DNA damage in a single cell can be determined, thereby evaluating the sample's ability to repair DNA damage.

[0062] Zebrafish were randomly selected and placed in 6-well plates, 30 fish per well. A zebrafish DNA damage model was established by water-soluble application of hydrogen peroxide. The sample groups were given water-soluble extract of *Saussurea involucrata* callus tissue at a weight percentage of 0.006%. A blank control group (without hydrogen peroxide) and a model control group (with hydrogen peroxide, but without *Saussurea involucrata* callus tissue extract) were also set up, with a volume of 3 mL per well. The liquid was changed daily, and the samples were incubated at 28°C in the dark for 120 h. Zebrafish samples were collected, ground, digested with trypsin, and stained by electrophoresis using a comet assay kit. After staining, 10 zebrafish cells were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using comet analysis software. The olivetail moment (D) value of zebrafish cell DNA was analyzed, and the DNA damage repair effect of the samples was calculated and determined according to the following formula: .

[0063] Table 11 Effects of Saussurea longifolia callus extract on DNA damage Project Name Sampling concentration Cellular DNA olive tail moment value Blank control - 1.842 Model control group (hydrogen peroxide stimulation) - 30.810 Snow lotus callus extract 0.006% 5.182 The results showed that, compared with the model control group, the DNAolive tail moment value of the callus extract of Saussurea longifolia was significantly reduced at a concentration of 0.006%, indicating that it has a certain repair effect on DNA damage.

[0064] The results of the above soothing and repair efficacy tests show that the extract of Saussurea longifolia callus tissue can promote the repair of skin cells from UV damage, promote ATP synthesis, reduce the movement distance of zebrafish, inhibit the expression of inflammatory factors in zebrafish, and repair DNA damage, demonstrating the efficacy of Saussurea longifolia callus tissue extract in anti-inflammatory, soothing, and repairing effects.

[0065] 3. Whitening effects of snow lotus callus extract (a) Effects of Saussurea longifolia callus extract on tyrosinase activity and melanin production in B16 cells Recent studies have shown that pigmentation is caused by ultraviolet radiation activating melanin-producing enzymes within melanocytes in the epidermis, leading to pigment production. In the human epidermis, melanocytes located in the basal layer contact and interact with surrounding keratinocytes, forming "epidermal melanin units." Melanin, a nitrogen-containing complex, is synthesized in melanosomes. Generally, the quantity and quality of melanin are crucial factors determining skin color. During melanin production, tyrosinase converts tyrosine into dopa and dopaquinone, which are then polymerized through non-enzymatic oxidation to form large melanin molecules. Therefore, inhibiting tyrosinase activity can limit melanin production and improve skin pigmentation. Additionally, skin color originates from the melanin stored within keratinocytes. Generally, people with more stored melanin have darker skin and are more protected from sun radiation. Tyrosinase is a complex copper-containing oxidoreductase widely found in microorganisms, animals, plants, and humans. In humans, tyrosinase is a key enzyme in the synthesis of melanin in the skin. Therefore, we used this model to evaluate the skin-whitening effect of the samples by detecting the content of tyrosinase and melanin in B16 cells.

[0066] The inhibitory effect of the samples on tyrosinase was determined using the L-Dopa oxidation method. Mouse melanoma B16 cells were inoculated at a concentration of 1×10⁻⁶. 5 Density culture was performed in 96-well plates. After 24 hours, the sample powder of Nagano snow lotus callus extract was dissolved in PBS, filtered for sterilization, added to cells, and cultured for 48 hours. The culture medium was removed, and 100 μL of PBS buffer containing 1% Triton X-100 was added to each well. Then, 50 μL of 0.2 mg / mL L-DOPA was added, and the cells were treated at 37°C for 3 hours before measuring the absorbance at 490 nm.

[0067] Enzyme activity was calculated using the following formula: Tyrosinase inhibition rate = [1 - (OD value of experimental group / OD value of control group)] × 100%. The effect of the samples on B16 cell viability was simultaneously determined using the MTT assay. Additionally, mouse melanoma B16 cells were cultured overnight at a density of 6000 in 96-well plates. After removing the supernatant, different concentrations of samples were added and cultured in medium containing 100 μM forskolin for 48 h. The supernatant was discarded, and the cells were treated with a solution containing the sample, 100 μM forskolin, and 100 μM dopa for 24 h. The absorbance at 405 nm was then measured.

[0068] Melanin inhibition rate = [1 - (OD value of experimental group / OD value of control group)] × 100%. Arbutin at a weight percentage of 0.01% was used as a positive control group. The results are shown in Tables 12 and 13.

[0069] Table 12 Effects of Saussurea longifolia callus extract on tyrosinase activity in B16 cells

[0070] Table 13 Effects of Saussurea longifolia callus extract on melanin production in B16 cells Project Name Test concentration Melanin inhibition rate (%) Blank control - 0 Positive control (arbutin) 0.01% 15 Snow lotus callus extract 0.015% 31 The results showed that the extract of Saussurea longifolia callus tissue could inhibit its tyrosinase activity and melanin production at a concentration of 0.0003% to 0.015% by weight.

[0071] (b) Inhibitory effect of Saussurea longifolia callus extract on tyrosinase activity and melanin production in zebrafish. Zebrafish are transparent in their early development. Melanin begins to grow from the retinal epithelium at 24 hours of embryonic development. Pigment cells originate from a group of cells differentiated from the dorsal ectoderm—neural crest cells—which then proliferate, migrate, and differentiate into melanocytes. Intervention during melanin formation can inhibit melanin production. Zebrafish tyrosinase activity assay: The whitening efficacy of *Saussurea involucrata* callus extract was tested using the standard "T / HPCIA 005-2021 Determination of Whitening Efficacy in Cosmetics—Zebrafish Embryo Method for Evaluating Whitening Efficacy". Zebrafish were randomly selected and placed in 6-well plates, 30 fish per well. 0.03% (w / w) of *Saussurea involucrata* callus extract was administered in water, with a blank control group included. Each well contained 3 mL of the extract. Three biological replicates were performed. The plates were incubated at 28 ℃ in the dark for 45 h. After collection, the samples were homogenized using a grinder, centrifuged, and the supernatant was collected. The protein concentration of each experimental group was determined using a BCA protein concentration assay kit. Take 250 μg of total protein from a zebrafish sample and add 1 mM levodopa solution at a volume ratio of 1:1, then mix well. Transfer the liquid to a 96-well plate, 200 μL / well, and measure the absorbance at 475 nm using a microplate reader. Calculate the whitening effect of the sample according to the formula; the inhibition rate of the sample on zebrafish tyrosinase is the whitening effect.

[0072] Table 14 Effects of Saussurea longifolia callus extract on tyrosinase activity Project Name Sampling concentration Tyrosinase activity inhibition rate (%) Blank control - 0 Snow lotus callus extract 0.03% 28 As shown in Table 14, the callus extract of Saussurea longifolia at a concentration of 0.03% inhibited tyrosinase activity, indicating that it has a certain whitening effect.

[0073] The skin-whitening effect of samples was evaluated by detecting the melanin content in zebrafish. Zebrafish melanin content detection: 30 zebrafish were randomly selected into 6-well plates, one well per zebrafish; samples were administered in water solution, with a blank control group included, and the volume per well was 3 mL. Three biological replicates were performed; incubation was carried out at 28 ℃ in the dark for 45 h. Samples were collected in 1.5 mL centrifuge tubes, each tube was added with RIPA lysis buffer, homogenized using a grinder, and the supernatant was discarded by centrifugation. 350 μL of 0.2 M NaOH solution was added to each tube, and the mixture was incubated at 60 ℃ for 1 h. A melanin standard solution was prepared using 0.2 M NaOH solution at a specific concentration. The standard solution and the sample to be tested were transferred to 96-well plates, 100 μL / well, and the absorbance (OD) was measured at 405 nm using a microplate reader. 405 Then, the melanin content (C) of each sample group is obtained according to the standard curve. The inhibition rate of the sample on zebrafish melanin is the whitening effect.

[0074] Table 15 Effects of Saussurea longifolia callus extract on melanin content Project Name Sampling concentration Melanin content (μg / mL) Blank control - 37.9 Snow lotus callus extract 0.03% 28.1 As shown in Table 15, compared with the blank control, the melanin content of the *Saussurea longifolia* callus extract at a concentration of 0.03% was significantly reduced, indicating that the *Saussurea longifolia* callus extract has a certain whitening effect. The whitening effect test results show that the *Saussurea longifolia* callus extract can inhibit tyrosinase activity and melanin production in both the B16 cell model and the zebrafish model, indicating that the *Saussurea longifolia* callus extract has a certain whitening effect.

[0075] 4. Firming and anti-wrinkle effects of Saussurea longipes callus extract (a) Tightening Elastin is the main component of elastic fibers in skin tissue, providing structural support and protecting the skin from aging and sagging. Elastin is composed of two types of short peptides arranged alternately. eln1 , eln2 Zebrafish are responsible for encoding different peptides of elastin, which makes the skin firm and elastic. Zebrafish have elastin similar to that in humans. eln1 , eln2 ) gene. Therefore, by detecting eln1 or eln2 The relative expression level of genes can indicate whether a sample has a firming effect.

[0076] The firming efficacy of the raw materials was evaluated using the standard "T / ZHCA 015-2022 Evaluation of Firming Efficacy of Cosmetics - Zebrafish Juvenile Elastin Gene Relative Expression Method for Assessment of Firming Efficacy". Zebrafish were randomly selected and placed in 6-well plates, 30 fish per well. Water-soluble samples were administered, with a blank control group included. Each well contained 3 mL of the sample. Three biological replicates were performed. The samples were incubated at 28℃ in the dark for 24 h. Total RNA was extracted from the zebrafish in each experimental group, cDNA was synthesized, and q-PCR was used for detection. β-actin And the expression of the target gene. Using β-actin As an internal reference for gene expression, the relative RNA expression level of the target gene is calculated.

[0077]

[0078] Table 16. Effects of long-leaved snow lotus callus extract on... eln1 Effects of gene expression Project Name Sampling concentration Firming effect (relative gene expression level) Blank control group - 1.002 Snow lotus callus extract 0.03% 1.506 The results showed that the extract from the callus tissue of *Saussurea longifolia* at a concentration of 0.03% was effective. eln1 The relative expression level of the gene was significantly increased compared with the blank control group, indicating that the sample has a certain firming effect.

[0079] (b) Anti-wrinkle Skin growth, repair, nutrition, elasticity, and tension are all related to collagen. Its loss leads to decreased skin smoothness and wrinkles. In tetrapods, type I collagen is a trimer, mainly composed of two α1 chains and one α2 chain, which are... col1a1a and col1a2 These genes encode collagen-related biological functions in connective tissue and bone. Three type I collagen genes exist in zebrafish, encoding the α1(I), α2(I), and α3(I) chains, respectively. col1a1a , col1a1b and col1a2 Therefore, by detection col1a1a or (and) col1a1b or (and) col1a2 The relative expression level of genes can indicate whether a sample has anti-wrinkle effects.

[0080] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. Water-soluble samples were administered, and a blank control group was also included. The volume per well was 3 mL. Three biological replicates were performed. The plates were incubated at 28°C in the dark for 24 h. Total RNA was extracted from the zebrafish in each experimental group, cDNA was synthesized, and detection was performed using q-PCR. β-actin And the expression of the target gene. Using β-actin As an internal reference for gene expression, the relative RNA expression level of the target gene was calculated. The detection results are shown in Table 17.

[0081]

[0082] Table 17. Effects of long-leaved snow lotus callus extract on... col1a1a Effects of relative gene expression Project Name Sampling concentration Relative gene expression level Blank control group - 1.004 Snow lotus callus extract 0.03% 1.461 The results showed that the extract from the callus tissue of Saussurea longipes at a concentration of 0.03% was effective. col1a1a The relative gene expression level was significantly increased compared to the blank control group, revealing that the sample has anti-wrinkle effects. The results of the four anti-wrinkle and firming related efficacy tests showed that the extract from the callus tissue of *Saussurea longipes* can promote… eln1 Genes and col1a1a Gene expression reflects its efficacy in firming and anti-wrinkle.

[0083] 5. Moisturizing effects of Saussurea longifolia callus extract (a) The effect of long-leaved snow lotus callus extract on aqp3a Effects of relative gene expression Zebrafish treated with sodium chloride will experience water loss and wrinkling of the skin surface due to osmotic pressure. Hyaluronic acid (HA) is present in the dermis and has moisturizing functions. Aquaporins (AQPs) are proteins located on cell membranes, forming "channels" that control the movement of water in and out of the cell. AQP-3, or aquaporin 3, is primarily an epidermal aquaporin, a membrane transport protein for water and glycerol, expressed in the plasma membrane of keratinocytes in the basal layer of the epidermis of normal skin, promoting water permeability and stratum corneum hydration. Therefore, by detecting... aqp3a The relative expression level of genes can indicate whether a sample has a moisturizing effect.

[0084] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. Water-soluble *Saussurea longifolia* callus samples (0.03% by weight) were administered. A blank control group and a model control group were also included, with a volume of 3 mL per well. Three biological replicates were performed. A zebrafish skin dehydration model was established by water-soluble administration of sodium chloride. The samples were incubated at 28°C in the dark for 22 h. Total RNA was extracted from the zebrafish in each experimental group, cDNA was synthesized, and detection was performed using q-PCR. β-actin And the expression of the target gene. Using β-actin As an internal reference for gene expression, the relative RNA expression level of the target gene is calculated.

[0085]

[0086] Table 18. Effects of long-leaved snow lotus callus extract on... aqp3a Effects of relative gene expression Project Name Sampling concentration Relative gene expression level Blank control - 2.706 Model control group (sodium chloride stimulation) - 1.002 Snow lotus callus extract 0.03% 2.819 The results showed that the extract from the callus tissue of Saussurea longipes at a concentration of 0.03% was effective. aqp3a The relative expression level of the gene was significantly increased compared with the model control group, indicating that the sample has a certain moisturizing effect.

[0087] (b) Effect of Saussurea longifolia callus extract on promoting hyaluronic acid synthesis Hyaluronic acid (HA) exists in the extracellular matrix between cells, acting as a filler and possessing moisturizing properties. It is found in both the epidermis and dermis. When skin is chronically dehydrated, the water content in skin cells and between cells decreases, leading to a reduction in hyaluronic acid levels. The space filled by the gel-like matrix, primarily composed of hyaluronic acid, shrinks, resulting in denser cell arrangement, collagen dehydration and hardening, and ultimately, aging of the stratum corneum. This causes the skin to appear rough, dry, and lacking elasticity. Zebrafish epidermis has a certain osmotic pressure tolerance range; exceeding this range leads to dehydration, causing the skin surface to shrink and reducing its hyaluronic acid content. Therefore, detecting hyaluronic acid content can indicate whether a sample has moisturizing effects.

[0088] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. Water-soluble *Saussurea longifolia* callus samples (0.03% by weight) were administered. A blank control group and a model control group were also included, with a volume of 3 mL per well. Three biological replicates were performed. Except for the blank control group, all experimental groups were given water-soluble sodium chloride to establish a zebrafish skin dehydration model. After incubation at 28℃ in the dark for 22 h, the samples were collected. After the experiment, zebrafish samples were collected, homogenized, centrifuged, and the supernatant was collected. Data were acquired using a zebrafish hyaluronic acid (HA) quantitative detection kit and an ELISA reader to analyze the hyaluronic acid content in the zebrafish and determine whether the samples had moisturizing effects.

[0089] Table 19 Effects of Saussurea longifolia callus extract on hyaluronic acid synthesis project Detection concentration Hyaluronic acid content (ng / mL) Blank control - 215 Model control group (sodium chloride stimulation) - 178 Snow lotus callus extract 0.03% 208 The results showed that the extract of Saussurea longifolia callus tissue at a concentration of 0.03% contained more hyaluronic acid than the model control group, indicating that the sample had a certain moisturizing effect.

[0090] Based on the results of the 5. Moisturizing-related efficacy tests, the extract of Saussurea longipes callus tissue can promote... aqp3a The expression of genes and the promotion of hyaluronic acid synthesis demonstrate its moisturizing effect.

[0091] Acne-fighting effects of extracts from the callus tissue of Saussurea longifolia. Acne is a series of inflammatory reactions triggered by the skin's inability to properly excrete sebum. It is a chronic inflammatory skin condition occurring in the hair follicle and sebaceous gland tissue, primarily affecting areas with high sebum secretion such as the face and chest / back. The clinical manifestations of acne vary, including mild comedones, papules, nodules, cysts, and even scars. Propionibacterium acnes is closely related to the development of acne, playing a specific role in promoting the release of inflammatory mediators. Propionibacterium acnes induces an inflammatory response in zebrafish, causing neutrophils to initiate an immune response, migrate to the skin epidermis, and aggregate. Neutrophils in the transgenic green fluorescent neutrophil strain of zebrafish (MPX) can be clearly observed under a fluorescence microscope; therefore, the efficacy of a sample in treating acne can be evaluated by observing real-time changes in the number of neutrophils in the zebrafish skin.

[0092] 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*. The sample was administered in water solution, with a blank control group and a model control group included; the volume per well was 3 mL. The plates were incubated at 28℃ in the dark for 3 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 number of neutrophils in the zebrafish was analyzed to determine whether the sample had acne-reducing effects. The results are shown in Table 20.

[0093] Table 20 Acne-fighting efficacy of Saussurea longifolia callus extract. Project Name Sampling concentration neutrophil count Blank control - 9.2 Model control group (induced by Propionibacterium acnes) - 26.9 Snow lotus callus extract 0.03% 19.6 The results in Table 20 show that the number of neutrophils in the 0.03% concentration of Saussurea longifolia callus extract was significantly reduced compared with the model control group, indicating that the sample has a certain acne-removing effect.

[0094] Sleep-improving effects of extracts from the callus tissue of 7-leaved Saussurea longifolia (1) Effects of longleaf snow lotus callus extract on melatonin receptor gene ( mtnr1aa The impact of expression Low doses of pentylenetetrazole (PTZ) can inhibit the synthesis and release of gamma-aminobutyric acid (GABA), causing brain neurons to lose their inhibitory function and become excitable, thus leading to insomnia. Melatonin is one of the hormones secreted by the pineal gland in the brain, which can induce natural sleep, and its secretion has a distinct circadian rhythm. At night, the activity of enzymes that synthesize melatonin in the pineal gland increases, and the level of melatonin secretion increases. When melatonin binds to specific receptors, it initiates cell signal transduction, shortens the sleep latency and prolongs the sleep duration, thereby overcoming sleep disorders, improving circadian rhythm disorders and improving sleep quality. When a person is in a state of insomnia, the body's melatonin secretion decreases, and the melatonin receptor gene (… mtnr1aa The expression of ) is reduced. Therefore, it can be detected by zebrafish mtnr1aa The relative expression level of genes is used to evaluate whether a sample has a sleep-improving effect.

[0095] Zebrafish were randomly selected and placed in 6-well plates, with 30 fish per well. Water-soluble *Saussurea longifolia* callus samples (0.0015% by weight) were administered. A blank control group and a model control group were also included, with a volume of 3 mL per well. Three biological replicates were performed. The plates were incubated at 28°C in the dark for 24 h. Except for the blank control group, all experimental groups were given water-soluble PTZ to establish a zebrafish insomnia model.

[0096] After incubation at 28℃ for another 1 hour, zebrafish samples were collected, total RNA was extracted from zebrafish in each experimental group, cDNA was synthesized, and detection was performed using q-PCR. β-actin And the expression of the target gene. Using β-actin As an internal reference for gene expression, the relative expression level of the target gene RNA was calculated. The detection results are shown in Table 21.

[0097]

[0098] Table 21. Effects of long-leaved snow lotus callus extract on melatonin receptor gene ( mtnr1aa The impact of expression Project Name Sampling concentration Relative gene expression level Blank control - 3.916 Model control group (penetrating tetrazolium) - 1.009 Snow lotus callus extract 0.015% 3.415 The results in Table 21 show that the extract of Saussurea longifolia callus tissue at a concentration of 0.015%... mtnr1aa The relative expression level of the gene was significantly increased compared with the model control group, revealing that the callus tissue of Saussurea longifolia at this concentration has a sleep-improving effect. (b) Effect of Saussurea longifolia callus tissue extract on gabra1 gene expression Pentylenetetrazole (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 mammalian central nervous system. It inhibits the excitability of neurons in the brain, thereby maintaining brain homeostasis. Low-dose PTZ can inhibit the synthesis and release of GABA, causing neurons in the brain to become excitable and thus leading to insomnia. When experiencing insomnia, the body's GABA levels decrease, and the expression of the GABA receptor gene (gabra1) also decreases. Therefore, the relative expression level of the gabra1 gene in zebrafish can be used to evaluate whether a sample has a sleep-improving effect.

[0099] Zebrafish were randomly selected and placed in 6-well plates, 30 fish per well. 0.0015% (w / w) of *Saussurea longifolia* callus extract was administered in water. A blank control and a model control group were also included. Each well contained 3 mL of extract. Three biological replicates were performed. The plates were incubated at 28 ℃ in the dark for 24 h. Except for the blank control group, all experimental groups were given PTZ in water to establish a zebrafish insomnia model. After incubation at 28 ℃ for another h, zebrafish samples were collected, total RNA was extracted from each group, cDNA was synthesized, and q-PCR was used for detection. β-actin And the expression of the target gene. Using β-actin As an internal control for gene expression, the relative RNA expression level of the target gene was calculated. The blank group represents the gene expression level before treatment, and the model control group represents the gene expression level after stimulation without the addition of Saussurea involucrata extract.

[0100] Table 22 Effects of Saussurea longifolia callus extract on the relative expression level of the gabra1 gene. Project Name Sampling concentration Relative gene expression level Blank control - 1.747 Model control group (penetrating tetrazolium) - 1.001 Snow lotus callus extract 0.015% 2.049 The results showed that, compared with the model control group, the extract of Saussurea longifolia callus tissue at a concentration of 0.015% upregulated the relative expression of the gabra1 gene, indicating that it has a certain effect on improving sleep.

[0101] When a person is sleep-deprived or has insufficient sleep, their skin is prone to dehydration and excessive sebum secretion, leading to wrinkles, dullness, and age spots. This demonstrates that sleep is essential for maintaining healthy skin. According to the results of seven related tests, the extract from the callus tissue of *Saussurea longipes* can promote the growth of the melatonin receptor gene (…). mtnr1aa )and gabra1 The expression of this gene demonstrates its ability to improve sleep, suggesting potential applications in skincare products and sleep aids.

[0102] Antioxidant activity of callus extract from 8. Saussurea longipes 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.

[0103] Zebrafish were randomly selected and placed in 6-well plates, with 15 fish per well. A zebrafish oxidative stress model was established by water-soluble administration of menadione. A water-soluble extract of *Saussurea longifolia* callus tissue was administered at 0.00125% by weight. A blank control group and a model control group were also included, with a volume of 3 mL per well. The plates were incubated at 28℃ 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 of the zebrafish yolk sac to determine whether the samples had antioxidant effects.

[0104] Table 23 Effects of Saussurea longifolia callus extract on fluorescence intensity of zebrafish yolk sac. Project Name Sampling concentration Yolk sac fluorescence intensity (pixels) Blank control - 350264 Model control group (menaquinone) - 860654 Snow lotus callus extract 0.00125% 762619 The results showed that the fluorescence intensity of zebrafish yolk sacs at a concentration of 0.00125% of the Saussurea longifolia callus extract was significantly lower than that of the model control group, indicating that the sample at this concentration could effectively scavenge free radicals and revealing that the sample has antioxidant effects.

[0105] 9. Immune-enhancing effects of Saussurea longifolia 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.

[0106] Zebrafish of uniform development were selected and injected with vinorelbine, then placed in 6-well plates with 15 fish per well. The samples were administered in water solution, with a blank control group and a model control group included, each well containing 3 mL. 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, analyzing the fluorescence intensity (S) of macrophages in the zebrafish tail veins to determine whether the samples had an immune-enhancing effect.

[0107] Table 24 Effects of Saussurea longifolia callus extract on macrophage fluorescence intensity Project Name Sampling concentration Macrophage fluorescence intensity (pixels) Blank control group - 126151 Model control group (tartaric acid stimulation) - 88931 Snow lotus callus extract 0.03% 113018 As shown in Table 24 and Figure 2The results showed that, compared with the model control group, the fluorescence intensity of zebrafish tail vein macrophages was significantly enhanced at a concentration of 0.03% by the callus extract of Saussurea longifolia, indicating that it has an immune-enhancing effect and can be applied to skin care products and health foods.

[0108] The effects of extract from the callus tissue of 10-leaf snow lotus on preventing hair loss, scalp care, and reducing dark circles. Microcirculation and blood circulation are closely related. Blood pumped from the heart first enters the arteries and then the capillaries, where it circulates and exchanges substances. Blood circulation in the head is peripheral circulation, or microcirculation. Microcirculation is the foundation of human growth and development, playing a regulatory role in metabolism and balancing cellular nutrition. When microcirculation is impaired, nutrient supply and waste removal are affected, leading to scalp problems such as gray hair or hair loss. Therefore, stimulating blood circulation in the head is crucial for ensuring nutrient supply and improving microcirculation. This requires awakening the capillaries within the scalp, promoting blood flow, improving microcirculation, and ensuring that nutrients reach the scalp via the bloodstream. Improving microcirculation can also alleviate dark circles caused by slow blood flow and oxygen deficiency in the skin's veins, leading to an accumulation of metabolic waste products.

[0109] Skin microcirculation refers to the blood circulation between arterioles and venules. Blood flow analyzers can be used to detect the blood flow velocity in zebrafish samples, analyze the improvement in microcirculation, and thus evaluate whether the samples have a microcirculation-improving effect.

[0110] Zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. Water-soluble samples were administered, with a blank control group included. Each well contained 3 mL of the sample. The plates were incubated at 28°C in the dark for 24 h. Ten zebrafish from each experimental group were randomly selected and placed in a blood flow and heart rate analyzer for video recording and data acquisition. The intersegmental blood flow velocity (V) of the zebrafish was analyzed, and the effect of the sample on improving microcirculation was calculated and determined using a formula.

[0111] Table 25 Effects of Saussurea longifolia callus extract on intersegmental blood flow velocity. Project Name Sampling concentration Intersegmental vascular blood flow velocity (μm / s) Enhancement effect Blank control group - 365 - Snow lotus callus extract 0.03% 505 38% The results showed that the intersegmental blood flow velocity of the extract from the callus tissue of Saussurea longifolia at a concentration of 0.03% was significantly increased compared with the blank control group, revealing that the sample has the effect of improving microcirculation and indicating that it has certain efficacy in skin care, hair loss prevention and hair care, and scalp care.

[0112] In summary, the callus extract of Saussurea longifolia has excellent anti-aging, sleep-improving, and immune-enhancing effects. It also has multiple functions such as soothing and repairing, whitening, anti-inflammatory, firming and anti-wrinkle, moisturizing, acne-removing, antioxidant, anti-hair loss and hair care, and scalp care. It can be used as an active additive in skin care and hair care products and food.

[0113] The above are preferred embodiments of the present invention, used to illustrate the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing an extract from callus tissue of Saussurea longipes, characterized in that, The method includes the following steps: a) Culture of callus cells: Young shoots of *Saussurea longifolia* were taken and inoculated onto MS medium with 0.3 g of 2,4-D. ~ 0.7 mg / L + 6-BA 1.8 ~ Callus tissue was obtained by induction with 2.2 mg / L sucrose, 30 g / L sucrose, and 3.5 g / L gellan gum. In proliferation medium B5 / MS / SH+6-BA 0.5 ~ 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or B5 / MS + mT 0.8 ~ 1.2 mg / L + 2,4-D 0.3 ~ In a solution of 0.7 mg / L sucrose, 30 g / L sucrose, and 3.5 g / L gellan gum, inoculate 1% of the proliferation medium volume. ~ 2% of the callus cells were cultured for proliferation to obtain the *Saussurea longifolia* callus cells; b) Preparation of extract from callus tissue of Saussurea longifolia: The callus cells of the long-leaved snow lotus were added to water for extraction to obtain the long-leaved snow lotus callus extract.

2. The preparation method according to claim 1, characterized in that, The induction medium consisted of MS + 2,4-D 0.5 mg / L + 6-BA 2.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, and the proliferation medium consisted of... ： B5 / MS / SH + 6-BA 0.5 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or B5 / MS + mT 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. The callus culture conditions are: temperature 20±1℃, humidity 50%~70%, 12 hours of light per day, and light intensity 1500 Lx ~ 2000 Lx.

3. The preparation method according to claim 1 is characterized in that, The preparation of the long-leaved snow lotus callus extract includes the following steps: A. Weigh the callus cells of the long-leaved snow lotus and grind them into pieces; B. Add 10g of long-leaved snow lotus callus cells. ~ Immerse and stir with 30 times the volume of deionized water, preferably 20 times the volume; C, 550 ~ Ultrasonic extraction at 650W, preferably 600W, followed by centrifugation and filtration. The supernatant was then filtered, and the filtrates were combined. D. Drying to obtain the long-leaved snow lotus callus extract.

4. An extract of *Saussurea longifolia* callus tissue obtained by the preparation method described in claims 1-3, characterized in that, The total polyphenol content in the *Saussurea longifolia* callus was 13%. ~ 23 mg / g, flavonoid content is 16 ~ 28mg / g, protein content is 168 ~ 178mg / g.

5. The long-leaved snow lotus callus extract as described in claim 4, characterized in that, The total polyphenol content of the *Saussurea longifolia* callus was 18.1 mg / g, the flavonoid content was 21.5 mg / g, and the protein content was 173 mg / g.

6. The use of the long-leaved snow lotus callus extract as described in claim 4 in the preparation of skin topical agents, hair topical agents, oral beauty agents, health products, food or food additives with anti-aging, soothing and repairing, whitening, anti-inflammatory, firming and anti-wrinkle, moisturizing, acne removal, sleep improvement, anti-oxidation, immune enhancement, anti-hair loss and hair care and scalp care effects.

7. The application as described in claim 6, characterized in that, The anti-aging effects include promoting the proliferation of human dermal fibroblasts, inhibiting β-galactosidase activity, promoting telomerase activity, promoting superoxide dismutase (SOD) activity, and increasing glutathione (GSH) content. The repair effects include repairing skin damage from UV exposure and increasing ATP content, thus repairing DNA damage. The soothing effects include reducing irritation caused by acetic acid. The anti-inflammatory effects include inhibiting the expression of inflammatory factors such as TNF-α, IL-1α, IL-6, and IL-8. The whitening effects include inhibiting tyrosinase activity and melanin production in human epidermal melanocytes (B16). The firming and anti-wrinkle effects include increasing the expression of the elastin eln1 gene and the col1a1a gene. The moisturizing effects include promoting the expression of the aqp3a gene and promoting hyaluronic acid synthesis. The acne-reducing effects include reducing neutrophil content. The sleep-improving effects include promoting the expression of the melatonin receptor gene mtnr1aa and GABA. The receptor gene gabra1 is expressed; the immune-enhancing effect is to increase macrophage activity; the hair loss prevention and scalp care effects are to enhance blood microcirculation.

8. A topical skin agent, characterized in that, The content of the long-leaved snow lotus callus extract in the aforementioned topical skin preparation is 0.001%. ~ The percentage of 1% refers to the mass of the *Saussurea longifolia* callus extract relative to the total mass of the topical skin agent. Preferably, the content of the *Saussurea longifolia* callus extract is 0.01%. ~ 1%.

9. A cosmetic oral preparation, characterized in that, The content of the long-leaved snow lotus callus extract in the aforementioned oral cosmetic preparation is 0.001%. ~ The percentage of 1% refers to the mass of the *Saussurea longifolia* callus extract relative to the total mass of the oral cosmetic preparation. Preferably, the content of the *Saussurea longifolia* callus extract is 0.01%. ~ 1%.

10. A health food product, characterized in that, The content of the long-leaved snow lotus callus extract in the aforementioned health food product is 0.001%. ~ The percentage of 1% refers to the mass of the *Saussurea longifolia* callus extract relative to the total mass of the skin health food product. Preferably, the content of the *Saussurea longifolia* callus extract is 0.01%. ~ 1%.

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