Sanabis lanceolata callus extract as well as preparation method and application thereof

Abstract

The invention relates to an application of a snow rabbit callus extract in the aspect of preparing a skin external preparation, a beautifying oral preparation, a health care product, food or a food additive, and is characterized in that the snow rabbit callus extract has better anti-inflammatory, soothing and whitening effects, and can be used for preparing the skin external preparation, the beautifying oral preparation, the health care product, the food or the food additive. Meanwhile, the composition also has the effects of repairing, tightening, resisting wrinkles, resisting oxidation, promoting autophagy, preserving moisture, removing acnes, resisting aging, enhancing immunity, resisting saccharification and the like, and can be applied to skin and hair care products and health-care foods as an active additive. The method has the advantages that the cells of the calluses of the snow rabbits are used as raw materials, the method has the advantages of infinite proliferation, stable quality of each batch, 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 laniceps Hand.-Mazz. callus extract and a preparation method and application thereof. BACKGROUND

[0002] Saussurea laniceps Hand.-Mazz. is also called Saussurea laniceps Hand.-Mazz. and is a perennial herb of the Compositae Saussurea DC. Its flowering and fruiting period is from August to October, and its leaves and capitula are covered with thick and fluffy hairs. It is distributed in Sichuan, Yunnan and Tibet of China and mainly grows in alpine scree at an altitude of 3,200-5,280 meters and is a typical alpine plant in China.

[0003] At present, Saussurea laniceps Hand.-Mazz. is still in a wild state and is a rare plant protected by the state, and no one has successfully planted Saussurea laniceps Hand.-Mazz. This greatly limits the development and utilization of the rare plant. In addition, there is no report on the research and application of Saussurea laniceps Hand.-Mazz. and its callus extract in skin care, hair care and health food. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a Saussurea laniceps Hand.-Mazz. callus extract preparation method and application thereof.

[0005] In one aspect, the present application provides a Saussurea laniceps Hand.-Mazz. callus extract preparation method, wherein the method comprises the following steps: a) culture of callus cells, taking the sprouts of Saussurea laniceps Hand.-Mazz. and inoculating them into an induction medium MS + 6-BA 0.3-0.7 mg / L + NAA 0.1-0.5 mg / L + sucrose 30 g / L + gelling gum 3.5 g / L to induce callus; inoculating callus with a volume of 1%-2% of a value-added medium into a value-added medium MS + 6-BA 0.3-0.7 mg / L + NAA 0.3-0.7 mg / L + potato powder 30 g / L + sucrose 30 g / L + gelling gum 3.5 g / L for value-added culture to obtain Saussurea laniceps Hand.-Mazz. callus cells; b) preparation of Saussurea laniceps Hand.-Mazz. callus extract: adding the Saussurea laniceps Hand.-Mazz. callus cells into water for extraction to obtain the Saussurea laniceps Hand.-Mazz. callus extract.

[0006] Further, the induction medium is MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, the proliferation medium is MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L, and the callus culture condition is temperature 20±1℃, humidity 50%-70%, and dark culture.

[0007] Further, the extraction of the callus of the E. prainii includes the following steps, A. The callus cells of the E. prainii are weighed and grinded; B. 10 ~ 30 volumes of deionized water, preferably 20 volumes of deionized water, are added for immersion and stirring; C. Ultrasonic extraction is performed at 550 ~ 650W, preferably 600W, centrifugal filtration is performed, the supernatant is taken for filtration, and the filtrates are combined; D. Drying is performed to obtain the callus extract of the E. prainii.

[0008] In another aspect of the present application, the callus extract of the E. prainii prepared by the preparation method is provided, and the total polyphenol content in the callus of the E. prainii is 15 ~ 25mg / g, the flavonoid content is 20 ~ 30mg / g, and the protein content is 240 ~ 250mg / g.

[0009] Further, the total polyphenol content in the callus of the E. prainii is 20.12mg / g, the flavonoid content is 26.60mg / g, and the protein content is 244.69mg / g.

[0010] The present application further provides the application of the callus extract of the E. prainii in the preparation of skin external agents, hair care external agents, beauty oral agents, health products, foods or food additives with the effects of whitening, soothing, anti-inflammatory, repairing, removing dark circles, tightening and anti-wrinkling, antioxidation, promoting autophagy, moisturizing, acne removal, anti-aging, enhancing immunity, anti-glycation, hair care and hair fixing.

[0011] Further, the whitening effect is to inhibit tyrosinase activity and melanin protein production of human epidermal melanocyte B16; the soothing effect is to inhibit expression of TNF-α, IL-1α, IL-6 and IL-8 inflammatory factors; the firming and anti-wrinkle effect is to promote expression of elastin eln1 gene and col1a1a; the moisturizing is to promote aqp3a gene expression and / or hyaluronic acid synthesis; the repair effect is to repair ultraviolet damaged cells and promote ATP and / or DNA damage repair; the anti-acne effect is to reduce neutrophil content; the immune enhancement effect is to increase macrophage activity; the anti-aging effect is to promote human dermal fibroblast proliferation, inhibit β-galactosidase activity, promote telomerase activity, promote superoxide dismutase (SOD) activity, and promote glutathione (GSH) content; the hair care and hair fixing effect is to enhance skin microcirculation, the antioxidant is to scavenge DPPH free radicals and / or reduce oxidative stress intensity; and the autophagy effect is to promote LC3-Ⅱ protein expression.

[0012] The application further provides a skin external agent, wherein the content of the Ochotona daurica callus extract in the skin external agent is 0.001% to 1%, and the percentage refers to the mass percentage of the Ochotona daurica callus extract in the total mass of the skin external agent.

[0013] The application further provides a beauty oral agent, wherein the content of the Ochotona daurica callus extract in the beauty oral agent is 0.001% to 1%, and the percentage refers to the mass percentage of the Ochotona daurica callus extract in the total mass of the beauty oral agent.

[0014] The application further provides a health food, wherein the content of the Ochotona daurica callus extract in the health food is 0.001% to 1%, and the percentage refers to the mass percentage of the Ochotona daurica callus extract in the total mass of the health food.

[0015] Beneficial effects The application obtains the callus of Ochotona daurica by using the method of plant cell tissue culture, and obtains the callus extract after extraction, and the effective component content of the callus extract is more excellent than that of wild medicinal material extract.

[0016] The Mian Tou Xue Lian callus extract has the advantages of sustainability, green environmental protection, stable quality and excellent effect, and solves the problems of wild plant resources of Mian Tou Xue Lian and difficult development and utilization.

[0017] The Mian Tou Xue Lian callus extract obtained by the application has the advantages of sustainability, green environmental protection, stable quality and excellent effect, and solves the problems of wild plant resources of Mian Tou Xue Lian and difficult development and utilization. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The Mian Tou Xue Lian callus plant medicinal material callus cells are arranged from left to right as a plant, a callus and a medicinal material.

[0019] Figure 2 Example two (3) comparison of soothing test results (in the figure, the red line represents the fast motion distance, the green line represents the medium speed motion distance, and the black line represents the slow speed motion distance).

[0020] Figure 3 Example two (10) comparison of fluorescence intensity of zebra fish yolk sac in the antioxidant test. DETAILED DESCRIPTION

[0021] The application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be pointed out that for those skilled in the art, without departing from the concept of the application, a number of changes and improvements can be made. These all belong to the protection scope of the application.

[0022] 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 application are commercially available.

[0023] Example one culture and extraction of Mian Tou Xue Lian callus

[0024] Take one of MS, B5, White, Heller or SH medium, cooperate with different plant growth regulators and cytokinins, and prepare the medium according to a certain ratio. Each adds 30g / L of sucrose and 3.5g / L of gelling agent, adjusts the pH to 5.7-5.8, sterilizes in a sterilization pot at 121℃ for 20min, and is ready for use. The culture conditions of all plant explants are 20±1℃, humidity 50%-70%, and dark culture.

[0025] The basic medium MS (M519) and plant growth regulators are purchased from Pytotechnolagy company. The plant growth regulators include: Naphthaleneacetic acid (NAA)NAA); 6-benzylaminopurine (6-BA).

[0026] 2. Disinfection of explants and culture of aseptic seedlings The seeds of wild Ochotona hyperborea were trimmed and washed under running water for 20 min, then washed with sterile water for 5 times. Then, the seeds were 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) at 110 rpm for 10 min in a shaker, washed with sterile water for 5 times, and then placed on MS basal medium at 13°C in the dark. When the seeds germinated and elongated to about 1 cm, the germination rate was counted and the aseptic seedlings were transferred to MS culture bottles for culture.

[0027] 3. Induction and proliferation of callus In the sterile clean bench, the leaves of the aseptic seedlings were cut into segments of 0.5-1.0 cm with a scalpel, and inoculated into the induction medium: MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + kappa carrageenan 3.5 g / L. The induced callus was cultured in the proliferation medium: MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + kappa carrageenan 3.5 g / L, and cultured for 28-30 days to obtain O. hyperborea callus cells with a multiplication coefficient of 4.

[0028] 4. Preparation of extracts and detection of active substances (1) Preparation of extracts The callus of O. hyperborea and the wild medicinal material of the aboveground part of O. hyperborea were ground and added with water (1:10 of solid-liquid ratio), and then extracted by ultrasonic (600 W) for 30 min. The mixture was centrifuged at 1000 rpm for 1 h, and the filtrate was obtained. Two types of sample solutions, callus extract and wild medicinal material extract, were obtained for subsequent testing of the contents of polyphenols, flavonoids and proteins in the extracts.

[0029] (2) Determination of total polyphenols The total polyphenol content was detected by Folin-Ciocalteu method. The standard curve was prepared by diluting gallic acid solution with a concentration of 0.3300 mg / mL into 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, and 0.3300 mg / mL, respectively. 100 μL of sample solution was mixed with 500 μL of 10% Folin-Ciocalteu reagent, and then allowed to stand for 5 min (not more than 8 min). Then, 400 μL of 7.5% Na2CO3 solution was added, and the mixture was allowed to stand at room temperature for 60 min. Finally, 100 μL of the mixture was measured for absorbance at 765 nm on a microplate reader. The total polyphenol content of the callus and wild medicinal material of M. tashiroi was calculated according to the standard curve. The results are shown in Table 1: the total polyphenol content of the callus of M. tashiroi was 20.12 mg / g, and the total polyphenol content of the wild medicinal material was 8.08 mg / g.

[0030] (3) Total flavone determination The total flavone content was detected by aluminum nitrate colorimetry. The standard solution was prepared by diluting quercetin in ethanol to a concentration of 0.2315 mg / mL, and then diluting it into 2-10 times of the concentration gradient. 300 μL of sample solution was mixed with 50 μL of 5% NaNO2 solution, and then allowed to stand at room temperature for 6 min. Then, 50 μL of 10% Al(NO3)3 solution was added, and the mixture was allowed to stand at room temperature for 6 min. Finally, 500 μL of 4% NaOH and 140 μL of deionized water were added, and the mixture was allowed to stand at room temperature for 15 min. The absorbance at 500 nm of 100 μL of the mixture was measured on a microplate reader. The total polyphenol content of the callus and wild medicinal material of M. tashiroi was calculated according to the standard curve. The results are shown in Table 1: the flavone content of the callus of M. tashiroi was 26.60 mg / g, and the flavone content of the wild medicinal material was 14.18 mg / g.

[0031] (4) Total protein determination The protein concentration of the sample was detected by using the Biyun Tian BCA Protein Assay Kit. The standard and working solutions were prepared according to the instructions, 100 μL of sample was added and mixed, and then allowed to stand at 60°C for 20-30 min. Finally, the absorbance at 562 nm was detected on a microplate reader, and the protein concentration of the sample was calculated according to the standard curve and the volume of the sample used. The results are shown in Table 1: the total protein content of the callus of M. tashiroi was 244.69 mg / g, and the total protein content of the wild medicinal material was 91.95 mg / g.

[0032] Table 1. Content of active ingredients in extracts Item Total polyphenol (mg / g) Total flavonoids (mg / g) Total protein (mg / g) Mian Tou Xue Rabbit Tissue 20.12 26.60 244.69 Mian Tou Xue Rabbit Wild Medicine 8.08 14.18 91.95 In summary, the callus of the snow rabbit has more polyphenols, flavonoids, and protein components than the wild medicinal material.

[0033] Take 1 mL of snow rabbit callus extract, use LC-MS test, get its extract contains syringin, ligustrazine, plant sphingosine and other active ingredients.

[0034] Example two: efficacy experiment 1. The repair effect of snow rabbit callus extract (1) The effect of repairing ultraviolet damaged cells Skin aging is divided into endogenous aging and exogenous aging, and sunlight, especially ultraviolet (ultraviolet UV) irradiation, is the main factor in the formation of exogenous aging, so exogenous aging is also called photoaging. The main UVA (320nm-400nm) and UVB (280-320nm) are related to skin photoaging. Many data show that ultraviolet radiation can induce fibroblast apoptosis, mitochondrial DNA deletion changes and increase of intracellular reactive oxygen free radicals, so we use UVB to irradiate human immortalized epidermis (Hacat) and human fibroblasts (FB) to establish a skin photo-damage model, and use the model to evaluate the effect of the sample in repairing and resisting photoaging.

[0035] Human immortalized epidermis (Hacat) cells and human fibroblasts (FB) were seeded in 96-well plates at 12000 cells / well and 6000 cells / well respectively, and cultured in a 5% CO2 cell incubator at 37°C for 24 hours. Then the cells were cultured in the medium containing the sample for 24 hours, and then the medium was replaced with PBS, and then UVB irradiation was used, with a dose of 40mJ / cm 2 .

[0036] The results are shown in Table 2.

[0037] Table 2. Effect of snow rabbit callus extract on ultraviolet radiation damage to skin cells Item Name Test Concentration Hacat Cell Viability (%) FB Cell Viability (%) Blank Control - 100 100 Model Control Group (UVB Radiation) - 41 72 Mian Tou Xue Rabbit Tissue Extract 0.01% 43 79 Mian Tou Xue Rabbit Medicine Extract 0.01% 35 89 Mian Tou Xue Rabbit Tissue Extract 0.03% 55 96 Mian Tou Xue Rabbit Medicine Extract 0.03% 37 24 Mian Tou Xue Rabbit Tissue Extract 0.05% 41 103 Mian Tou Xue Rabbit Medicine Extract 0.05% 21 26 Snow rabbit callus extract at 0.01%-0.05% concentration has obvious protective effect on UVB-induced human Hacat cell damage, and the effect is better than that of snow rabbit medicinal material extract at the same concentration.

[0038] Snow rabbit callus extract at 0.03%-0.05% concentration has protective effect on UVB-induced human fibroblasts, and the effect is better than that of snow rabbit medicinal material extract at the same concentration.

[0039] (2) The effect of repairing DNA damage Comet assay, also known as single cell gel electrophoresis assay, 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 of cells, 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, while under the action of alkaline electrolyte, DNA is de-spiraled, and damaged DNA breaks and fragments are released. Because these DNA has small molecular weight and is alkaline denatured into single-stranded DNA, the negatively charged DNA will migrate from the nuclear DNA to the positive electrode to form a "comet" image during electrophoresis, while the undamaged DNA part remains spherical. The more serious the DNA damage, the more 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 and damage bases, so by measuring the length of the migrated DNA, the degree of DNA damage in a single cell can be measured, and the repair effect of the sample on DNA damage can be evaluated.

[0040] Randomly selected zebrafish in 6-well plates, 30 per well. Hydrogen peroxide was given to establish a zebrafish DNA damage model. The sample was given in water, and a normal control group and a model control group were set up, with a volume of 3 mL per well. The liquid was replaced once a day, and the incubation was carried out at 28°C in the dark for 120 h. The zebrafish samples were collected, ground and added with trypsin for digestion, and then electrophoresis and staining were carried out using a comet assay kit. After staining, 10 zebrafish cells were randomly selected from each experimental group and placed under a fluorescence microscope for photography. 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 was calculated whether the sample had DNA damage repair effect, and the calculation formula was as follows:

[0041] The results are shown in Table 3.

[0042] Table 3. Effect of Mian Tou Xue Lu Zi Callus Extract on DNA Damage Repair Item Name Test Concentration DNA Olive Tail Moment (Tail Distance) Value (D) DNA Damage Repair Effect (%) Normal Control Group - 1.84 - Hydrogen Peroxide DNA Damage Model Control Group - 30.8 - Mian Tou Xue Rabbit Tissue Extract 0.01 9.18 75 Compared with the model control group, the DNA olivetail moment value of 0.01% detection concentration of L. leucantha callus extract was significantly reduced, indicating that it had a certain repair effect on DNA damage. Therefore, L. leucantha callus extract promotes skin cell UV damage repair; at the same time, it can reduce the degree of DNA damage, and has a good damage repair effect.

[0043] 2. Whitening effect of L. leucantha callus extract The "T / ZHCA 012-2021 Test Method for Whitening Effect of Cosmetics on Zebrafish Embryos" was used to evaluate the whitening effect of L. leucantha callus extract.

[0044] Zebrafish is transparent throughout the early 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, then proliferate, migrate, and differentiate into pigment cells. Intervention during the process of melanin formation can inhibit the formation of melanin.

[0045] (1) Tyrosine activity detection 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 are generated. Therefore, the whitening effect of the sample can be evaluated by the activity of tyrosinase.

[0046] Zebrafish tyrosinase activity detection experiment: randomly select zebrafish in a 6-well plate, 30 per well; water-soluble sample is given, and a 0.05% L. leucantha callus extract solution is prepared, 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 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 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:

[0047] As shown in Table 4, Table 4. Effect of snow rabbit head callus extract on tyrosinase activity Item Name Test Concentration OD475 Whitening Effect (%) Normal Control Group - 0.087 - Mian Tou Xue Rabbit Tissue Extract 0.05 0.053 39 At a concentration of 0.05% by mass, the tyrosinase inhibitory effect of snow rabbit head extract was measured to be optimal.

[0048] (2) Melanin protein content detection The melanin protein content of zebrafish was detected to evaluate whether the sample had whitening efficacy. Zebrafish melanin protein content detection: randomly selected zebrafish in a 6-well plate, 30 per well; water-soluble sample was given, and a snow rabbit head callus extract with a mass percentage of 0.05% was prepared, 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 an enzyme marker to measure the absorbance value (OD 405 ) at 405 nm, and then obtain the melanin protein content (C) of each sample group according to the standard curve. The inhibition rate of the sample on zebrafish melanin protein is the whitening efficacy, which is calculated according to the formula and judged whether the sample has whitening efficacy, the calculation formula is as follows:

[0049] As shown in Table 5, at a concentration of 0.05% by mass, the snow rabbit extract was detected to have a melanin protein inhibitory effect.

[0050] Table 5. Effect of snow rabbit head callus extract on melanin protein content Item Name Test Concentration Melanin Protein Content (C) Whitening Effect (%) Normal Control Group - 37.9 - Mian Tou Xue Rabbit Tissue Extract 0.05 14.6 61 As can be seen from the above, at a concentration of 0.05% by mass, the snow rabbit head callus extract can effectively inhibit tyrosinase activity and melanin protein production, and has optimal whitening efficacy.

[0051] 3. Anti-inflammatory and soothing efficacy of snow rabbit head callus extract (1) Exercise distance monitoring Glacial acetic acid has strong piercing and corrosive properties. The skin will have a stinging and burning sensation when it comes into contact with glacial acetic acid. Zebrafish with incomplete 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, so that the more the movement trajectory is, the more serious the pain is. The behavior analyzer is used to detect the total movement distance of zebrafish, and whether the sample has soothing effect is evaluated.

[0052] Randomly select zebrafish in a 6-well plate, 1 tail per well. The sample is dissolved in water to prepare a 0.05% mass percentage of mian tou xue rabbit callus extract solution, and a normal control group and a model control group are set up, with a volume of 3 mL per well. Incubate at 28°C in the dark for 24 hours. Randomly select 10 zebrafish from each experimental group and transfer them to a 96-well plate. Glacial acetic acid is used to establish a zebrafish pain model. Immediately use a behavior analyzer to measure the movement trajectory of zebrafish, analyze and collect the total movement distance (D) of zebrafish, and calculate and determine whether the sample has soothing effect according to the formula.

[0053]

[0054] Table 6. Soothing effect of mian tou xue rabbit callus extract Item Name Test Concentration Total Movement Distance (D) Relieving Effect (%) Normal Control Group - 115 - Glacial Acetic Acid Pain Model Control Group - 5662 - Mian Tou Xue Rabbit Tissue Extract 0.05 4110 39 The results are shown in Table 6. Compared with the model group, the total movement distance of zebrafish in the 0.05% mian tou xue rabbit callus extract solution was significantly reduced, indicating that it had a certain soothing effect.

[0055] (2) Reducing the expression of inflammatory factors Sodium dodecyl sulfate (SLS) can cause the body to produce a stimulating response when it acts on the body. The stimulant enters the zebrafish body, induces an inflammatory response, and the neutrophils respond to the immune response, migrate to the skin epidermis and aggregate. TNF- α is 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 mononuclear cells and vascular endothelial cells, and its level rises rapidly when inflammation occurs. The relative expression of zebrafish TNF-α , IL-1α , IL-6 and IL-8 genes can be detected to evaluate whether the sample has soothing effect.

[0056] Randomly select zebrafish in 6-well plates, 30 per well. Water-soluble administration of SLS to establish zebrafish skin inflammation model. Water-soluble administration of samples, while setting up a blank group, model control group, 3 mL per well. Three biological replicates. 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 using q-PCR β-actin and target genes. Use β-actin as the internal reference for gene expression, and calculate the 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 stimulus without adding the extract of the callus of the rabbit ear.

[0057] As shown in Table 7, compared with the model group, the extract of the callus of the rabbit ear significantly down-regulated the relative expression of TNF-α , IL-1α , IL-6 and IL-8 genes, indicating that it has good anti-inflammatory and soothing effects.

[0058] Table 7. Effect on the relative expression of TNF-α , IL-1α , IL-6 and IL-8 genes Item Name Test Concentration Normal Control Group SLS Inflammatory Model Control Group Mian Tou Xue Rabbit Tissue Extract 0.05 0.211 1 0.701 0.05 0.227 1 0.771 0.05 0.258 1 0.216 0.05 0.239 1 0.845 In summary, 0.05% of the extract of the callus of the rabbit ear significantly reduced the expression of TNF-α , IL-1α , IL-6 and IL-8 inflammatory factors caused by sodium dodecyl sulfate (SLS), and had better anti-inflammatory and soothing effects.

[0059] 4. The extract of the callus of the rabbit ear improves microcirculation, removes dark circles under the eyes, and protects and strengthens hair Microcirculation is closely related to blood circulation. Blood output by the heart first enters each arterial blood vessel, and then enters capillaries for blood circulation and material exchange. Blood circulation in the head belongs to peripheral circulation, i.e., microcirculation, which plays a role in regulating metabolism and balancing cell nutrition. When microcirculation function is impaired, nutrient supply and waste removal will be affected. For hair, it will cause problems such as white hair or hair loss. Therefore, stimulating blood circulation in the head is crucial for ensuring nutrient supply to the head and activating microcirculation. 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. At the same time, improving microcirculation can slow down dark circles under the eyes caused by too slow blood flow in skin veins, poor blood circulation, and lack of oxygen leading to accumulation of too much metabolic products.

[0060] Microcirculation refers to the blood circulation between the arterioles and the venules. The blood flow speed of the test zebrafish is detected by using a blood flow analyzer, and the improvement of microcirculation is analyzed to evaluate whether the sample has the effect of improving microcirculation.

[0061] Randomly select zebrafish in a 6-well plate, 15 per well. The sample is given in water, and the mass percentage of the mian tou xue rabbit callus extract is 0.05%. A normal control group is set up, and the volume of each well is 3 mL. 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), and calculate and judge whether the sample has the effect of improving microcirculation according to the formula.

[0062]

[0063] The results are shown in Table 8. The intersegmental blood vessel blood flow speed of the sample mian tou xue rabbit callus extract is significantly increased compared with the normal control group, revealing that the sample has the effect of improving skin microcirculation.

[0064] Table 8. Effect of mian tou xue rabbit callus extract on improving microcirculation Item Name Test Concentration Blood Flow Velocity (V) Improving Microcirculation Effect (%) Normal Control Group - 365 - Mian Tou Xue Rabbit Tissue Extract 0.05 507 39 Therefore, the mass percentage of 0.05% of the mian tou xue rabbit callus extract can increase the intersegmental blood vessel blood flow speed and improve the effect of microcirculation by 39%, significantly improve the blood microcirculation, and have the application prospect of removing dark circles and protecting hair and firming hair.

[0065] 5. Firming effect of mian tou xue rabbit callus extract The firming effect of mian tou xue rabbit callus extract is evaluated by using "T / ZHCA 015-2022 Evaluation of the firming effect of cosmetics on zebrafish larvae by elastin gene relative expression amount method". 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 which are responsible for encoding different peptide segments of elastin, and the two genes together regulate the expression level of elastin to make the skin firm and elastic. Zebrafish has similar elastin ( eln1 、 eln2 ) genes as humans. Therefore, by detecting the relative expression amount of eln1 or (and) eln2 gene, it can be shown whether the sample has the firming effect.

[0066] Randomly select zebrafish in 6-well plates, 30 per well. Water-soluble administration of samples, preparation of 0.05% of the mass percentage of the extract of the callus of the rabbit, and set up a normal control group, 3 mL per well. Three biological replicates. Incubate at 28°C for 24 hours in the dark. Extract total RNA from zebrafish in each experimental group, synthesize cDNA, and use q-PCR to detect β- actin gene expression. Use β-actin as the internal reference for gene expression, and calculate the relative expression of the target gene.

[0067]

[0068] The results are shown in Table 9 Table 9. Tightening efficacy of the extract of the callus of the rabbit Item Name Test Concentration Gene Relative Expression Normal Control Group - 1 Mian Tou Xue Rabbit Tissue Extract 0.05 2.74 Therefore, the extract of the callus of the rabbit with a mass percentage of 0.05% significantly increased the expression of the elastin gene eln1 , and had a tightening effect.

[0069] 6. Autophagy-promoting effect of the extract of the callus of the rabbit Autophagy is a self-protection mechanism that is ubiquitous in eukaryotic cells. Cells have aging and damaged organelles, incorrectly synthesized proteins, invading bacteria, and macromolecular substances, etc. Autophagy encapsulates these substances that are harmful to cells in autophagosomes, and combines with lysosomes to form autolysosomes to decompose and remove these substances, maintain the homeostasis of the intracellular environment, and recycle intracellular substances. Protein LC3 plays a key role in the autophagy process. LC3 exists in two forms. When cellular autophagy is formed, the cytoplasmic form (LC3-I) is enzymatically digested into a small polypeptide, and is converted into the membrane form (LC3-II). Therefore, the increase in the ratio of LC3-II / β-actin can be used to determine whether the autophagy level is increased.

[0070] Randomly select 4-day-old zebrafish in 6-well plates, 30 per well. Water-soluble administration of 0.01% of the mass percentage of the extract of the callus of the rabbit, and set up a normal control group, 3 mL per well. Three biological replicates. Incubate at 28°C for 24 hours in the dark. After incubation, use RIPA lysis buffer to lyse the cells and extract the protein. Use Western Blot to detect β-actin and the expression of LC3-II protein. The ratio of LC3-II / β-actin is the relative expression of LC3-II protein. The higher the relative expression of LC3-II protein, the better the autophagy.

[0071] The results are shown in Table 10, Table 10. Effect of the extract of the callus of the rabbit zokor on the expression of LC3-Ⅱ protein Item Name Test Concentration LC3-Ⅱ Protein Relative Expression Promoting Autophagy Effect (%) Normal Control Group - 0.543 - Mian Tou Xue Rabbit Tissue Extract 0.01 0.831 53 Therefore, compared with the blank control group, the relative expression of LC3-Ⅱ protein of the zebrafish administered with 0.01% of the extract of the callus of the rabbit zokor was significantly increased, indicating that the extract of the callus of the rabbit zokor has the effect of promoting autophagy, which can achieve the effects of removing metabolic waste of the skin and delaying aging.

[0072] 7 Anti-wrinkle effect of the extract of the callus of the rabbit zokor The growth, repair, nutrition, elasticity and tension of the skin are related to collagen, and the loss of collagen will make the skin smoothness decrease 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, respectively, and performs collagen-related biological functions in connective tissue and bone. There are three type I collagen genes in zebrafish, which encode α1 (I), α2 (I) and α3 (I) chains, respectively. col1a1a , col1a1b and col1a2 Therefore, by detecting the relative expression of col1a1a or (and) col1a1b or (and) col1a2 genes, it can be indicated whether the sample has the anti-wrinkle effect.

[0073] Randomly select zebrafish in a 6-well plate, 30 fish per well. Water administration of 0.05% of the extract of the callus of the rabbit zokor, and set 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.

[0074]

[0075] The results are shown in Table 11, Table 11. Anti-wrinkle effect of the extract of the callus of the rabbit zokor Item Name Test Concentration Gene Relative Expression Normal Control Group - 1 Mian Tou Xue Rabbit Tissue Extract 0.05 1.74 Therefore, compared with the normal control group, the relative expression of col1a1a gene of the zebrafish administered with 0.05% of the extract of the callus of the rabbit zokor increased by 74%, revealing that the sample has the anti-wrinkle effect.

[0076] 8 Moisturizing effect of Hylotelephium villosum callus extract (1) Effect on aqp3a Relative expression of genes Zebrafish treated with sodium chloride will lose water and shrink due to osmotic pressure. Hyaluronic acid (HA) exists in the dermis of the skin and has a moisturizing function. Aquaporins (AQPs) are proteins located on the cell membrane, which form "pores" on the cell membrane and can control the entry and exit of water in cells. AQP-3, i.e. water channel protein 3, is mainly an epidermal water channel protein, 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 can indicate whether the sample has moisturizing effect.

[0077] Randomly selected zebrafish in 6-well plates, 30 per well. Water-soluble administration of 0.05% Hylotelephium villosum callus extract sample, while setting up normal control group and model control group, 3 mL per well. Three biological replicates. 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 zebrafish in each experimental group, synthesize cDNA, and detect β-actin Gene expression of target genes. Use β-actin As the internal reference for gene expression, calculate the RNA relative expression of the target gene.

[0078]

[0079] The results are shown in Table 12 Table 12. Moisturizing effect of Hylotelephium villosum callus extract Item Name Test Concentration Gene Relative Expression Normal Control Group - 2.71 Sodium Chloride Water Deficit Model Control Group - 1.00 Mian Tou Xue Rabbit Tissue Extract 0.05 1.55 0.05% Hylotelephium villosum callus extract can increase aqp3a The relative expression of genes, compared with the model control group, significantly increased, revealing that the sample has potential moisturizing effect.

[0080] (2) Effect on promoting hyaluronic acid synthesis Hyaluronic acid (HA) exists in the extracellular matrix between cells, and is a filler between cells, which has a moisturizing function. Hyaluronic acid is contained in the epidermis and dermis. When the skin is long-term dry and dehydrated, the water content of skin tissue cells and intercellular water decreases, the content of hyaluronic acid in the skin decreases, the space filled by the gel matrix composed of hyaluronic acid between cells decreases, which leads to close arrangement of cells, dehydration and hardening of collagen, causing keratin layer aging, making the skin rough, dry and losing elasticity. The zebrafish epidermis has a certain osmotic pressure tolerance range, and dehydration will occur when the osmotic pressure exceeds the tolerance range, resulting in skin surface dehydration and shrinkage and a decrease in the content of hyaluronic acid in the body. Therefore, the content of hyaluronic acid can indicate whether the sample has a moisturizing effect.

[0081] Randomly select zebrafish in a 6-well plate, 30 per well. Water-soluble administration of 0.05% of the mass percentage of the extract of the callus of the rabbit zonalasmius thibetanus sample, and set up a normal control group and a model control group, with a volume of 3 mL per well. Three biological repeats. Except for the normal control group, the rest of the experimental groups were water-soluble administered with sodium chloride to establish a zebrafish skin dehydration model. After incubation in the dark at 28°C for 22 h. After the experiment, collect the zebrafish samples in 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 content of hyaluronic acid (C) in the zebrafish, calculate and judge whether the sample has a moisturizing effect according to the formula, and the calculation formula is as follows:

[0082] The results are shown in Table 13.

[0083] Table 13. Effect of the extract of the callus of the rabbit zonalasmius thibetanus on the efficacy of hyaluronic acid Item Name Test Concentration Hyaluronic Acid Content (C) Moisturizing Effect (%) Normal Control Group - 215 - Sodium Chloride Water Deficit Model Control Group - 178 - Mian Tou Xue Rabbit Tissue Extract 0.05 202 65 As can be seen from the above, the extract of the callus of the rabbit zonalasmius thibetanus with a mass percentage of 0.05% can promote the synthesis of hyaluronic acid, and has a moisturizing effect.

[0084] 9. Acne-removing effect of the extract of the callus of the rabbit zonalasmius thibetanus 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, etc. The clinical manifestations of acne are not the same, including mild acne, papules, nodules, cysts and even scars, etc. 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 inflammatory response in zebrafish, neutrophil immune response, migration and aggregation to the epidermis of the skin. The neutrophils of transgenic zebrafish (MPX) can be observed under a fluorescence microscope, so the efficacy of the sample can be evaluated by observing the skin of the zebrafish in real time. Change in Neutrophil Number

[0085] Randomly selected zebrafish in a 6-well plate, 15 per well. Injected with propionibacterium acnes to establish an inflammatory model in zebrafish. Water-soluble administration of 0.05% of the sample of the callus extract of the rabbit zokor, and set up a normal control group and a model control group, with a volume of 3 mL per well. Incubate at 28°C in the dark for 3 h. Randomly select 10 zebrafish from each experimental group and place them under a fluorescence microscope for photography. Use advanced image processing software to analyze and collect data, analyze the number of neutrophils (N) in zebrafish, and calculate and determine whether the sample has acne-removing efficacy according to the formula.

[0086]

[0087] The results are shown in Table 14.

[0088] Table 14. Acne-removing efficacy of the callus extract of the rabbit zokor Item Name Test Concentration Neutrophil Number (N) Acne-Removing Effect (%) Normal Control Group - 9.20 - Propionibacterium Acnes Inflammatory Model Control Group - 26.9 - Mian Tou Xue Rabbit Tissue Extract 0.05 19.6 41 Therefore, the number of neutrophils in the callus extract of the rabbit zokor was significantly reduced compared with the model control group, indicating that the callus extract of the rabbit zokor can reduce the inflammatory response caused by propionibacterium acnes, revealing the potential of the sample to have acne-removing efficacy.

[0089] 10. Antioxidant effect of the callus extract of the rabbit zokor Radical scavenging effect of the callus extract of the rabbit zokor ​1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable nitrogen-centered organic free radical. The DPPH method was proposed in 1958 and is widely used to measure the antioxidant capacity of biological samples, classification substances and food. This method is based on the fact that DPPH free radical has a single electron, which has a strong absorption at 517 nm, and its alcohol solution is purple. When there is a free radical scavenger, its absorption gradually disappears due to pairing with its single electron, and the degree of discoloration is quantitatively related to the number of electrons it receives, so it can be used to quickly and quantitatively analyze the fluorescence intensity of the sample by spectrophotometer to detect the free radical scavenging condition, so as to evaluate the antioxidant capacity of the sample.

[0090] 0.1 mL of the extract sample solution was added to a test tube, 0.1 mL of DPPH ethanol solution was added, mixed, and reacted in the dark at room temperature for 30 min. The OD value was measured at 525 nm, and the clearance rate was calculated according to the following formula: clearance rate I (%) = [1-(T-T0) / (C-C0)]x100%, wherein: T0: absorbance of 0.1 mL sample liquid plus 0.1 mL 95% ethanol; T: absorbance of 0.1 mL sample liquid plus 0.1 mL DPPH solution; C0: absorbance of 0.1 mL water plus 0.1 mL 95% ethanol; C: absorbance of 0.1 mL water plus 0.1 mL DPPH solution.

[0091] The results are shown in Table 15.

[0092] Table 15. DPPH clearance rate of L. yunnanensis callus extract Item Name Test Concentration (mg / mL) DPPH Scavenging Rate (%) ​ 1 92.57 Extract of callus of rabbit foot snow 0.5 44.10 Therefore, the L. yunnanensis callus extract has a certain free radical scavenging effect.

[0093] CellROX® reagent is a DNA dye, which is weakly fluorescent by itself, can be oxidized by intracellular oxygen free radicals (ROS), and the oxidation product binds to DNA to produce bright green fluorescence. CellROX® is soluble in organic solvents (such as dimethyl sulfoxide), and the main component of zebrafish yolk sac is fat, so CellROX® has strong permeability in the yolk sac, and the staining in this part is obvious. Menaquinone can produce reactive oxygen free radicals, and when the amount of free radicals produced is greater than the body's clearance capacity, oxidative stress occurs. The fluorescence intensity of zebrafish yolk sac is used to evaluate whether the sample has antioxidant effect.

[0094] Randomly select zebrafish in 6-well plates, 15 per well. Water-soluble administration of menadione to establish zebrafish oxidative stress model. Water-soluble administration of 0.001% of the sample of the callus extract of the roots of the rabbit plantain willow, while setting up a normal control group and a model control group, with a volume of 3 mL per well. Incubate at 28°C in the dark for 22 h. Stain the zebrafish with a specific ROS fluorescent reagent, and after the staining is complete, randomly select 10 zebrafish from each experimental group and place them under a fluorescence microscope to take pictures. Use advanced image processing software to analyze and collect data, analyze the fluorescence intensity (S) of the yolk sac of zebrafish, and calculate and determine whether the sample has antioxidant effects according to the formula.

[0095]

[0096] The results are shown in Table 16. Table 16. Antioxidant effects of the callus extract of the roots of the rabbit plantain willow Item name Detection concentration Yolk sac fluorescence intensity (S) Antioxidant effect (%) Normal control group - 2267694 - Menaquinone oxidative stress model control group - 4214407 - Extract of callus of rabbit foot snow 0.001 3898060 16 Compared with the model control group, the addition of 0.05% of the callus extract of the roots of the rabbit plantain willow can reduce the fluorescence intensity of the yolk sac of zebrafish due to oxidative stress, and has certain antioxidant effects. The immune system of zebrafish is highly conserved, and its immune cell types and morphology are similar to those of humans, with neutrophils, macrophages, and lymphocytes (B cells and T cells). Zebrafish, like mammals, have an innate immune system and an adaptive immune system, and the innate immune system is fully mature and functional at 48 h after fertilization (48 hpf). A large dose of vinorelbine tartrate can inhibit the formation of bone marrow, leading to a decrease in platelets, red blood cells, neutrophils, macrophages, and ultimately to low immunity. Therefore, by detecting the fluorescence intensity of macrophages in the tail vein of zebrafish, we can evaluate whether the sample has an immune-enhancing effect.

[0097] Pick zebrafish that are developing consistently, inject vinorelbine, and place them in 6-well plates, 15 per well. Water-soluble administration of 0.05% of the callus extract of the roots of the rabbit plantain willow, while setting up a normal control group and a model control group, with a volume of 3 mL per well. Incubate at 28°C in the dark for 48 h. Randomly select 10 zebrafish from each experimental group and place them under a fluorescence microscope to take pictures. Use advanced image processing software to analyze and collect data, analyze the fluorescence intensity (S) of the tail vein macrophages of zebrafish, and calculate and determine whether the sample has an immune-enhancing effect according to the formula, which is as follows:

[0098] The results are shown in Table 17.

[0099] Table 17. Effects of callus extract from Saussurea involucrata on immune-enhancing effects Item name Detection concentration Macrophage fluorescence intensity (S) Moisturizing efficacy (%) Normal control group - 126151 - Vinorelbine immunosuppression model control group - 88931 - Extract of callus of rabbit foot snow 0.05 105721 45 Compared with the model control group, the fluorescence intensity of macrophages in the zebrafish tail veins of the group containing 0.05% (w / w) of Saussurea costatus callus extract was significantly enhanced, indicating that it has an immune-enhancing effect.

[0100] 12. Anti-aging effects of callus extract from Saussurea costatum. (1) Effect on the proliferation of human dermal fibroblasts 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.

[0101] The prepared Saussurea involucrata callus extract samples were added to human fibroblast (FB) culture medium at mass percentages of 0.01%, 0.02%, and 0.04%, with a solvent without the sample serving as a blank control. After 48 hours of culture, the 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%. The effect on FB cell proliferation was calculated to evaluate the anti-aging effect of the sample.

[0102] The results are shown in Table 18. Table 18. Effects of callus extract from Saussurea involucrata on dermal fibroblast proliferation. Item name Test concentration FB cell viability (%) Blank control - 100 Extract of callus of rabbit foot snow 0.01% 134 Extract of callus of rabbit foot snow 0.02% 162 Extract of callus of rabbit foot snow 0.04% 172 The callus extract of Saussurea costatum can promote the proliferation of human dermal fibroblasts at a concentration of 0.01%-0.04%, indicating that the callus extract of Saussurea costatum has a certain anti-aging effect.

[0103] (2) β-galactosidase activity Peroxides entering the body generate free radicals, causing lipid peroxidation, which disrupts the stability of biological membranes, produces lipofuscin, damages nuclear and mitochondrial DNA, causes protein oxidative damage and macromolecular cross-linking, thereby inducing aging. β-galactosidase (SA-β-gal) is a marker of cellular aging; in senescent cells, SA-galactosidase activity and expression increase. Therefore, the average staining intensity of SA-galactosidase is used to evaluate whether a sample has anti-aging effects.

[0104] Randomly select zebrafish in 6-well plates, 15 per well. Hydrogen peroxide is administered in water to establish a zebrafish aging model. The zebrafish are administered 0.01% of the extract of the callus of the roots of the rabbit plantainwillow in water, and a normal control group and a model control group are set up, with a volume of 3 mL per well. The liquid is replaced once a day. Incubate in the dark at 28°C for 120 h. Fix the zebrafish with 4% cell tissue fixative overnight, and then use a β-galactosidase staining kit to perform staining. After staining overnight, randomly select 10 zebrafish from each experimental group and place them under a dissecting microscope to take photographs. Use advanced image processing software to analyze and collect data, analyze the β-galactosidase staining intensity values of the zebrafish, and the greater the value, the stronger the β-galactosidase activity.

[0105] The results are shown in Table 19.

[0106] Table 19. Effect of the extract of the callus of the roots of the rabbit plantainwillow on β-galactosidase activity Item name Detection concentration β-galactosidase staining intensity value Anti-aging effect (%) Normal control group - 0.195 - Hydrogen peroxide aging model control group - 0.261 - Extract of callus of rabbit foot snow 0.01 0.237 36 The average β-galactosidase staining intensity of zebrafish administered 0.01% of the extract of the callus of the roots of the rabbit plantainwillow was significantly reduced compared with the model control group, indicating that the extract of the callus of the roots of the rabbit plantainwillow has an anti-aging effect.

[0107] (3) Telomerase activity Free radicals are produced in the body by peroxides, causing lipid peroxidation and thus destroying the stability of biological membranes, producing lipofuscin, damaging DNA and mitochondrial DNA in the nucleus, causing protein oxidative damage and macromolecular crosslinking, and thus inducing aging. Telomeres have important biological functions, and as age increases, 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 gradually shorten with age, at which point telomerase plays a role in slowing the shortening of telomeres, 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 a sample has an anti-aging effect.

[0108] Randomly select zebrafish in 6-well plates, 30 per well. Hydrogen peroxide is administered in water to establish a zebrafish aging model. The zebrafish are administered 0.01% of the extract of the callus of the roots of the rabbit plantainwillow 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 replicates are performed. The liquid is replaced once a day. Incubate in the dark at 28°C for 120 h. Collect the samples, grind them to homogenate, centrifuge to obtain the supernatant, and then use a telomerase activity ELISA kit to perform determination. Use a microplate reader to collect data, analyze the OD450 values of the expression levels of telomerase activity in each experimental group, and calculate the telomerase activity from the OD450 values.

[0109] The results are shown in Table 20.

[0110] Table 20. Effect of L. japonicus callus extract on telomerase activity Item name Detection concentration Relative activity of telomerase Anti-aging effect (%) Normal control group - 9.10 - Hydrogen peroxide aging model control group - 6.44 - Extract of callus of rabbit foot snow 0.01 8.39 73 The telomerase activity of the zebrafish administered with 0.01% L. japonicus callus extract sample was significantly increased compared with the model control group, which can significantly delay telomere shortening, revealing that the L. japonicus callus extract with a mass percentage of 0.01% has an anti-aging effect.

[0111] (4) Superoxide dismutase (SOD) activity The aging of zebrafish has some similarities with the aging process of humans. Hydrogen peroxide induces DNA damage response through oxidative stress and activates the p53 / p21 signaling pathway, thereby leading to zebrafish aging. During oxidative stress, 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.

[0112] Randomly selected zebrafish in a 6-well plate, 30 per well. Hydrogen peroxide was administered in water to establish a zebrafish aging model. The L. japonicus callus extract sample with a mass percentage of 0.001% 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 repeats. The liquid was changed once a day. Incubate at 28°C in the dark for 120 h. After the experiment, collect the zebrafish samples in each group, grind the homogenate, centrifuge to obtain the supernatant, use the SOD activity detection kit, use the enzyme marker to collect data, and analyze the SOD activity in the body of zebrafish.

[0113] The results are shown in Table 21 Table 21. Effect of L. japonicus callus extract on superoxide dismutase (SOD) activity Item name Detection concentration Relative activity of SOD (U / mg prot) Anti-aging effect (%) Normal control group - 12 - Hydrogen peroxide aging model control group - 9 - Extract of callus of rabbit foot snow 0.001 10 26 The SOD activity of the zebrafish administered with 0.001% L. japonicus callus extract was significantly enhanced compared with the model control group, revealing that the sample has an anti-aging effect.

[0114] (5) Glutathione (GSH) content 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 aging of zebrafish. 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 content of GSH.

[0115] Randomly select zebrafish in a 6-well plate, 30 per well. Hydrogen peroxide is administered in water to establish a zebrafish aging model. The sample of the extract of the callus of Lepidogrammus neglevus is administered in water at a mass percentage of 0.001%, and a normal control group and a model control group are set up, with a volume of 3 mL per well. Three biological repeats. The liquid is replaced once a day. Incubate in the dark at 28°C for 120 h. After the experiment, collect the zebrafish samples in each group, grind and homogenize, centrifuge to take the supernatant, use the GSH content detection kit, use the enzyme label instrument to collect data, and analyze the GSH content in the body of zebrafish. The higher the GSH content, the better the anti-aging effect.

[0116] The results are shown in Table 22, Table 22. Effect of the extract of the callus of Lepidogrammus neglevus on the content of glutathione (GSH) Item name Detection concentration Glutathione (GSH) content (μmol / g prot) Anti-aging effect (%) Normal control group - 39 - Hydrogen peroxide aging model control group - 18 - Extract of callus of rabbit foot snow 0.001 24 27 The GSH content of the sample of the extract of the callus of Lepidogrammus neglevus is significantly increased compared with the model control group, revealing that the sample has an anti-aging effect.

[0117] In summary, the extract of the callus of Lepidogrammus neglevus can promote the proliferation of human dermal fibroblasts, promote the activity of telomerase and superoxide dismutase, promote the content of glutathione in zebrafish, and inhibit the activity of β-galactosidase, and has good anti-aging effect.

[0118] 13. Anti-glycation effect of the extract of the callus of Lepidogrammus neglevus Glycation is a non-enzymatic process that covalently binds free sugars to proteins. This process is not controlled by enzymes, so it is a non-enzymatic reaction. Glycation occurs spontaneously in the blood and irreversibly binds sugars or sugar degradation products to proteins. Since the French scientist Maillard discovered this reaction, it is also called the Maillard reaction. In addition, glycation is a process that destroys proteins, reducing their stability and functionality, so the glycation reaction interferes with normal physiological functions.

[0119] Glucose, fructose, or galactose is a sugar added in the glycation process. Through glycation, sugars only react with mature proteins. The first step of glycation is condensation, which is a time-consuming process since the reaction does not involve enzymes. The final product of condensation is an unstable Schiff base or ethylenediamine. Then, ethylenediamine spontaneously rearranges to form an Amadori product, which is a stable ketamine, and the product is further degraded. Aminoguanidine is commonly used as a positive control to inhibit the glycation reaction. Aminoguanidine is a drug for treating complications of diabetes and is an inhibitor that blocks the generation of carbonyl groups in intermediate molecules in the glycation reaction.

[0120]

[0121] The glycation reaction is one of the culprits that causes skin aging, laxity, wrinkles, and yellowing. Glycated collagen can be observed in the skin from the age of 20. As age increases, the proportion of glycation can reach 30-50% by the age of 80. In the anti-glycation evaluation system, by detecting various advanced glycation end products, the effect of the raw material on the complex multi-path glycation reaction can be inferred.

[0122] Prepare 90 mg / ml solutions of glucose, galactose, and fructose, respectively, and additionally add bovine serum albumin (BSA) to a final concentration of 20 mg / ml, and add 0.01% and 0.1% of the extract of the callus of the rabbit ear of the genus Lippia or 10 mM of aminoguanidine (positive control) to each reducing sugar-BSA solution and mix well, and add 100 ul of the mixture to each well of a 96-well plate and incubate at 37°C for 14 days. After 14 days of reaction, detect the fluorescence value of each well at an emission wavelength of 355 nm and an absorption wavelength of 460 nm.

[0123] The results are shown in Tables 23-25 below Table 23. Inhibition rate of glycation of the extract of the callus of the rabbit ear of the genus Lippia on the glucose-BSA system Item name Detection concentration Inhibition rate of glycation Positive control group 10 mM 34.31 Extract of callus of rabbit foot snow 0.1% 36.55 Extract of callus of rabbit foot snow 0.01% 4.6 Table 24. Inhibition rate of glycation of the extract of the callus of the rabbit ear of the genus Lippia on the galactose-BSA system Item name Detection concentration Inhibition rate of glycation Positive control group 10 mM 36.35 Extract of callus of rabbit foot snow 0.1% 36.62 Extract of callus of rabbit foot snow 0.01% 5.84 Table 25. Inhibition rate of sugarization of fructose-BSA system by callus extract of L. leucura Item name Detection concentration Inhibition rate of glycation Positive control group 10 mM 28.21 Extract of callus of rabbit foot snow Extract of callus of rabbit foot snow Item name Detection concentration Inhibition rate of glycation Positive control group 10 mM Extract of callus of rabbit foot snow 0.1% 13.6 From the above Tables 23-25, it can be seen that the callus extract of L. leucura with a mass percentage of 0.1% can better inhibit the sugarization of glucose-BSA system, the sugarization of galactose-BSA system and the sugarization of fructose-BSA system, and has an effect equivalent to that of 10 mM aminoguanidine, and can obviously delay the aging caused by skin sugarization.

[0124] In summary, the callus extract of L. leucura prepared by the present application contains rich flavonoids, polyphenols and proteins and other components, and has more contents than plant medicinal material extract, and has the effects of anti-inflammation, soothing, whitening, repairing, firming and anti-wrinkle, antioxidation, promoting autophagy, moisturizing, acne removal, anti-aging, enhancing immunity, anti-sugarization and the like, and can be applied in skin care and hair care products and health foods. The callus extract of L. leucura 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.

[0125] The above is the 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 shall be covered within the protection scope of the present application.

Claims

1. A method for preparing an extract from callus tissue of the snow rabbit, characterized in that, The method includes the following steps: a) Culture of callus cells Tender shoots of Saussurea costatum were taken and inoculated into MS induction medium containing 0.3–0.7 mg / L 6-BA, 0.1–0.5 mg / L NAA, 30 g / L sucrose, and 3.5 g / L gellan gum to induce callus formation. In MS medium containing 0.3–0.7 mg / L 6-BA, 0.3–0.7 mg / L NAA, 30 g / L potato starch, 30 g / L sucrose, and 3.5 g / L gellan gum, callus cells were inoculated at 1%–2% of the medium volume for proliferation culture to obtain the aforementioned Saussurea laniceps callus cells. b) Preparation of callus extract from Saussurea involucrata The callus cells of the snow rabbit were added to water for extraction to obtain the snow rabbit callus extract.

2. The preparation method according to claim 1, characterized in that, The induction medium consisted of MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. The proliferation medium consisted of MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + potato starch 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L. The callus culture conditions were: temperature 20±1℃, humidity 50%~70%, and dark culture.

3. The preparation method according to claim 1, characterized in that, The extraction of callus tissue from the Saussurea laniceps includes the following steps: A. Weigh the callus cells from the snow rabbit and grind them into pieces; B. Add 10g of Snow Rabbit 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 callus extract of the snow rabbit.

4. An extract of callus tissue from *Saussurea involucrata* obtained by the preparation method described in claims 1-3, characterized in that, The total polyphenol content in the callus tissue of the snow rabbit was 15%. ~ 25mg / g, flavonoid content is 20 ~ 30mg / g, protein content is 240 ~ 250mg / g.

5. The *Saussurea involucrata* callus extract as described in claim 4, characterized in that, The total polyphenol content of the *Saussurea involucrata* callus was 20.12 mg / g, the flavonoid content was 26.60 mg / g, and the protein content was 244.69 mg / g.

6. The application of the Snow Rabbit callus extract as described in claim 4 in the preparation of skin topical agents, hair care topical agents, oral beauty agents, health products, food or food additives with effects such as whitening, soothing, anti-inflammatory, repairing, dark circle removal, firming and anti-wrinkle, anti-oxidation, promoting autophagy, moisturizing, acne removal, anti-aging, enhancing immunity, anti-glycation, and hair care and strengthening.

7. The application as described in claim 6, characterized in that, The whitening effect is achieved by inhibiting the tyrosinase activity and melanin production of human epidermal melanocytes B16; the soothing effect is achieved by inhibiting the expression of inflammatory factors such as TNF-α, IL-1α, IL-6, and IL-8; the firming and anti-wrinkle effect is achieved by increasing the expression of elastin eln1 gene and col1a1a; the moisturizing effect is achieved by promoting aqp3a gene expression and / or hyaluronic acid synthesis; the repairing effect is achieved by repairing UV-damaged cells and promoting ATP enhancement and / or DNA damage repair; the acne-removing effect is achieved by reducing neutrophil content; the immune-enhancing effect is achieved by increasing macrophage activity; the anti-aging effect is achieved by promoting the proliferation of human dermal fibroblasts, inhibiting β-galactosidase activity, promoting telomerase activity, promoting superoxide dismutase (SOD) activity, and promoting glutathione (GSH) content; the hair-strengthening effect is achieved by enhancing skin microcirculation; the antioxidant effect is achieved by scavenging DPPH free radicals and / or reducing oxidative stress intensity; and the autophagy effect is achieved by increasing LC3-II protein expression.

8. A topical skin agent, characterized in that, The content of the *Saussurea involucrata* callus extract in the topical skin preparation is 0.001%. ~ The percentage of 1% refers to the percentage by mass of the *Saussurea involucrata* callus extract relative to the total mass of the topical skin agent; preferably, the content of the *Saussurea involucrata* callus extract is 0.01%. ~ 1%.

9. A cosmetic oral preparation, characterized in that, The content of the *Saussurea involucrata* callus extract in the oral cosmetic preparation is 0.001%. ~ The percentage of 1% refers to the mass of the *Saussurea involucrata* callus extract relative to the total mass of the oral cosmetic preparation; preferably, the content of the *Saussurea involucrata* callus extract is 0.01%. ~ 1%.

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

Images