Saussurea saluenensis cell culture extract as well as preparation method, application and skin care product composition thereof

Abstract

The invention relates to a saussurea saluenensis cell culture extract as well as a preparation method and application thereof and a skin care product composition, and the method comprises the following steps: S1, collecting saussurea saluenensis cell culture, and drying the saussurea saluenensis cell culture; s2, the material obtained in the step S1 is extracted with ethyl alcohol, the volume ratio of the material to the ethyl alcohol is 1: 5-1: 20, the extraction time is 4-8 hours, and the volume fraction of the ethyl alcohol is 60-80%; s3, separating the extracting solution and extracting residues in the step S1, and performing centrifugal treatment on the extracting solution to obtain centrifugate; and S4, concentrating the centrifugate to obtain a fluid extract. According to the method, the active substances are extracted from the cell culture of the saussurea saluenensis, so that the influence of external factors such as planting conditions, extraction parts and seasons on the activity of the extract can be avoided, the consistency and effectiveness of the extract are ensured, and a reliable basis is provided for the research of the saussurea saluenensis extract on the aspects of promoting collagen synthesis, resisting oxidation and the like.

Description

Technical Field

[0001] The present disclosure relates to the field of extraction of plant cell cultures, and particularly to an extract of Saussurea salwinensis cell cultures, a preparation method thereof, applications, and a skin care composition. Background Art

[0002] Collagen is a key protein for maintaining skin elasticity and structural integrity. As people age, the synthesis of collagen in the human body decreases, leading to skin aging and the appearance of wrinkles. Therefore, finding effective methods to promote collagen synthesis is of great significance for improving skin health. Oxidative stress is closely related to the occurrence of various skin problems, aging, and diseases. Antioxidants can protect cells by scavenging free radicals and reducing oxidative damage. In addition, antioxidants can also promote the synthesis of human collagen.

[0003] Traditionally, plant extracts have been widely studied for promoting collagen synthesis. As a plant of the genus Saussurea in the Compositae family, Saussurea salwinensis is rich in flavonoids and polyphenols like its related species Saussurea involucrata, and has antioxidant activity. However, the research on the antioxidant properties of Saussurea salwinensis extracts is relatively less at present. In addition, current plant extraction technologies mostly rely on extracting active ingredients from the whole plant or its specific parts. For example, only the active substances in the roots, stems, or leaves of the plant are extracted for analysis and research.

[0004] This method is not only restricted by the raw material supply, but also difficult to control the content and quality of the active ingredients. In addition, the active ingredients directly extracted from plants may fluctuate due to factors such as planting conditions and seasonal changes, affecting the consistency and effectiveness of the extracts. Summary of the Invention

[0005] The purpose of the present disclosure is to improve the consistency and effectiveness of Saussurea salwinensis extracts, thereby providing a reliable basis for its research in promoting collagen synthesis and antioxidant properties.

[0006] To achieve the above purpose, the present disclosure provides a preparation method of an extract of Saussurea salwinensis cell cultures, comprising the following steps:

[0007] S1. Collect Saussurea salwinensis cell cultures and perform a drying treatment;

[0008] S2. Extract the material obtained in step S1 with ethanol, the volume ratio of the material to the ethanol is 1:5 to 1:20, the extraction time is 4 to 8 hours, and the volume fraction of the ethanol is 60 to 80%;

[0009] S3. Separate the extract and the extraction residue in step S1, and perform centrifugation on the extract to obtain a centrifugate;

[0010] S4. Concentrate the centrifugate to obtain a fluid extract.

[0011] Preferably, in step S1, the Saussurea salwinensis cell culture is dried to a water content of ≤ 10%.

[0012] Preferably, in step S2, the volume ratio of the material to the ethanol is 1:10, the volume fraction of the ethanol is 70%, the single extraction time is 6 hours, and the extraction is carried out 3 times.

[0013] Preferably, in step S3, the extract and the extraction residue are separated successively through a sieve and a pipeline filter.

[0014] More preferably, in step S3, before the centrifugation treatment of the extract, the extract is concentrated, and the extract is concentrated to 20 - 30% of the original volume.

[0015] The present disclosure also provides an extract of Saussurea salwinensis cell culture, which is prepared by the preparation method described in any one of the foregoing.

[0016] The present disclosure also provides an application of an extract prepared by the preparation method described in any one of the foregoing in skin antioxidant.

[0017] The present disclosure also provides an application of an extract prepared by the preparation method described in any one of the foregoing in promoting skin collagen synthesis.

[0018] The present disclosure also provides a skin care product composition, which contains an extract of Saussurea salwinensis cell culture prepared by the preparation method described in any one of the foregoing.

[0019] Preferably, the addition amount of the extract of Saussurea salwinensis cell culture is 0.1% - 0.5%.

[0020] The technical solutions claimed by the present disclosure have achieved the following beneficial effects:

[0021] 1) Extracting active substances from Saussurea salwinensis cell culture can avoid the influence of external factors such as raw material planting conditions, extraction parts, and seasons on the activity of the extract, ensure the consistency and effectiveness of the extract, and provide a reliable basis for the research on Saussurea salwinensis extract in promoting collagen synthesis and antioxidant and other aspects.

[0022] 2) Using Saussurea salwinensis cell culture as a raw material and combining with the extraction method and parameters used in the present disclosure, an extract with a higher total flavonoid content and richer other active ingredients can be obtained.

[0023] 3) The extract obtained from the cell culture of Saussurea salwinensis shows more excellent antioxidant capacity and ability to promote collagen synthesis compared to the extract directly obtained from the Saussurea salwinensis plant, providing a stable basis for the application of the active substances of Saussurea salwinensis in the pharmaceutical and cosmetic industries. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only the embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the provided drawings.

[0025] Figure 1 Schematic diagram of the extraction process of the extract from the cell culture of Saussurea salwinensis.

[0026] Figure 2 Extraction efficiency of the extract from the cell culture of Saussurea salwinensis under different extraction conditions.

[0027] Figure 3 Ion chromatogram of LC-MS / MS mass spectrometry analysis of the extract from the cell culture of Saussurea salwinensis.

[0028] Figure 4 Shows the antioxidant capacity of the extract from the cell culture of Saussurea salwinensis.

[0029] Figure 5 Shows the expression levels of COL1A1 (A) and COL3A1 (B) genes in human skin fibroblasts under different treatment conditions.

[0030] Figure 6 Shows the determination results of the content of type I collagen in human skin fibroblasts under different treatment conditions. Detailed Embodiments

[0031] To make the objectives, technical solutions, and beneficial effects in the embodiments of the present disclosure clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present disclosure with reference to the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are some, but not all, of the embodiments of the present disclosure. Based on the embodiments of the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present disclosure.

[0032] Example 1

[0033] As Figure 1 shown, this embodiment provides a method for preparing an extract from the callus cell culture of Saussurea salwinensis, including the following steps:

[0034] 1. Collect the Saussurea salwinensis cell cultures that have been cultured for 21 days, wash them 3 times with deionized water, drain the water, and place them in an oven at 55°C for 12 hours to ensure that the water content is less than 10%;

[0035] 2. Use the UV method to measure the total flavonoid content in the dried product of the culture

[0036] The determination of total flavonoids is based on the method for the determination of total flavonoids in the "Technical Guidelines for the Inspection and Evaluation of Physical and Chemical and Hygienic Indicators of Health Foods (2020 Edition)". The detailed method is as follows:

[0037] A.1 Test instruments and reagents

[0038] Rutin reference substance, 5% sodium nitrite solution, 10% aluminum nitrate solution, 4% sodium hydroxide test solution, analytical pure methanol, analytical balance, ultraviolet-visible spectrophotometer

[0039] A.2 Test method

[0040] A.2.1 Preparation of reference substance solution

[0041] Precisely weigh 10 mg of rutin reference substance dried to constant weight at 120°C, place it in a 50 mL volumetric flask, dissolve it with methanol, and dilute it to the mark, then shake well.

[0042] A.2.2 Preparation of standard curve

[0043] Precisely pipette 1, 2, 3, 4, 5, and 6 mL of the reference substance solution into 25 mL volumetric flasks respectively, add water to each to 6 mL, add 1 mL of 5% sodium nitrite solution, shake well, let stand for 6 minutes, add 1 mL of 10% aluminum nitrate solution, shake well, let stand for 6 minutes, add 10 mL of 4% sodium hydroxide test solution, add water to the mark, shake well, let stand for 15 min. Using the corresponding reagent as the blank, according to the spectrophotometry (General Principles 0401, Volume IV, Chinese Pharmacopoeia 2020 Edition), measure the absorbance at a wavelength of 510 nm, and calculate the linear regression equation with the absorbance and its corresponding concentration (μg / ml). The equation obtained is: y = kx + b

[0044] A.2.3 Preparation of test solution

[0045] Take about 5 ml of this product, precisely weigh it, place it in a 50 ml volumetric flask, make up the volume with 60% methanol, shake well, then precisely pipette 5 ml, place it in a 50 ml volumetric flask, make up the volume with 60% methanol to the mark, shake well, and use it as the test solution.

[0046] A.2.4 Determination method

[0047] Precisely pipette 5 mL of the test solution, transfer it into a 25 mL volumetric flask, and operate according to the method described under the preparation of the standard curve, starting from "add water to 6 mL". Take the corresponding reagent as the blank, measure the absorbance at a wavelength of 510 nm, and calculate the amount of rutin in the test solution from the linear regression equation.

[0048] Content calculation formula:

[0049] X = (A 样 - b) × V 样 / (k * M 样 ) * 1000000 × 100%

[0050] Where: X - the percentage content of the total flavonoids contained in the sample

[0051] A 样 - the absorbance value measured for the sample

[0052] k, b - the slope and intercept in the standard curve

[0053] V 样 - the dilution factor during the sample extraction process (2500)

[0054] M 样 - the sampling amount of the sample during sample determination (g).

[0055] 3. Perform ethanol extraction, with a total of 27 combinations of extraction conditions set (as shown in Table 1). The solid-liquid ratio is set according to the mass of the dried culture and the volume of edible alcohol (meeting the national standard GB / T 10343 - 2023). A total of 3 solid-liquid ratios (material mass: reagent volume) are designed: 1:5, 1:10, and 1:20. The volume concentration of edible alcohol is set at 60%, 70%, and 80%. Extraction is carried out 3 times in total, and the extraction duration for each time is set at 4 hours, 6 hours, and 8 hours;

[0056] 4. Filter the extraction residue through a pipeline filter and the sieve at the bottom of the pipeline equipment in sequence, and collect the extract;

[0057] 5. The collected extract is concentrated to 1 / 4 of the original volume by vacuum concentration. At this time, the alcohol concentration is between 15% and 20%, and it is cooled to room temperature;

[0058] 6. Release the concentrated dilute alcohol solution in Step 5, centrifuge it with a tubular centrifuge, discard the centrifugation residue, and obtain the centrifugate;

[0059] 7. Further concentrate the centrifugate to a non-alcohol state under vacuum, with the temperature not exceeding 60 °C, to obtain a fluid extract.

[0060] Table 1

[0061]

[0062] As can be seen from the results in Table 1 and Figure 2 Under the extraction conditions where the ratio of the dry product of Saussurea salwinensis cell culture to edible ethanol is 1:10, the concentration of edible ethanol is 70%, the number of extraction times is 3, and the single extraction time is 6 hours, the extraction rate of the extract of Saussurea salwinensis cell culture is the highest, which is 83% (relative to the dry product of Saussurea salwinensis cell culture). The total flavonoid content of the obtained fluid extract (calculated as 1 ml of fluid extract corresponding to 1 g of dry product) is the highest, reaching 16.68%.

[0063] Example 2

[0064] In this example, LC-MS / MS mass spectrometry analysis was performed on the extract of Saussurea salwinensis cell culture obtained in Example 1:

[0065] Using an EXION LC System (SCIEX) ultra-high performance liquid chromatograph, chromatographic separation of the target compound was carried out through a Waters UPLC liquid chromatographic column. The mobile phase A was an aqueous solution containing 0.1% formic acid, and the mobile phase B was acetonitrile. The column oven temperature was 40 °C, the auto-sampler temperature was 4 °C, and the injection volume was 2 μL. Among them, the time unit was min, and the flow rate unit was μL / min.

[0066] 3. Using a SCIEX 6500QTRAP+ triple quadrupole mass spectrometer equipped with an Ion Drive Turbo V ESI ion source, mass spectrometry analysis was carried out in the multiple reaction monitoring (MRM) mode. The ion source parameters were as follows: Ion Spray Voltage: +5500 / -4500 V, Curtain Gas: 35 psi, Temperature: 400 °C, Ion Source Gas1: 60 psi, Ion Source Gas 2: 60 psi, DP: ±100 V.

[0067] All mass spectrometry data acquisition and quantitative analysis of the target compound were completed through the SCIEX Analyst WorkStation Software (Version 1.6.3). The original mass spectrometry was converted into TXT format using the MS conventer software. Then, the peak extraction, annotation, etc. were completed using the self-written R program package combined with the self-built database.

[0068] Figure 3 The LC-MS / MS mass spectrometry analysis ion chromatograms (EICs) of the extract sample of Saussurea salwinensis cell culture are shown.

[0069] Through comparative analysis, a total of 699 compounds were identified, including 205 shikimic acid and phenylpropionic acid substances including flavonoids, 130 terpenoids, 108 alkaloids, and 55 amino acids and small peptides.

[0070] In this example, 15 most enriched compounds are exemplarily listed (refer to Table 2), and the main components are as follows: caffeic acid, chlorogenic acid, 1,5-dicaffeoylquinic acid, 5-caffeoylshikimic acid, cryptochlorogenic acid, azelaic acid, syringin, chrysanthemin, coniferol glucoside, aesculetin, umbelliferone, γ-aminobutyric acid, arginine, and choline.

[0071] Table 2

[0072] Number Compound Molecular formula Retention time RT 1 Caffeic acid <![CDATA[C 9 H 8 O 4 > 246.377 2 Chlorogenic acid <![CDATA[C 16 H 18 O 9 > 218.13 3 1,5-Dicaffeoylquinic acid <![CDATA[C 25 H 24 O 12 > 224.007 4 5-Caffeoylshikimic acid <![CDATA[C 16 H 16 O 8 > 246.801 5 Cryptochlorogenic acid <![CDATA[C 16 H 18 O 9 > 223.079 6 Azelaic acid <![CDATA[C 9 H 16 O 4 > 304.785 7 Syringin <![CDATA[C 17 H 24 O 9 > 216.936 8 Patuletin <![CDATA[C 28 H 32 O 14 > 219.701 9 Coniferin <![CDATA[C 16 H 22 O 8 > 212.978 10 Fraxetin <![CDATA[C 10 H 8 O 5 > 229.334 11 Umbelliferone <![CDATA[C 9 H 6 O 3 > 276.114 12 γ-Aminobutyric acid <![CDATA[C 4 H 9 NO 2 > 48.631 13 Arginine <![CDATA[C 6 H 14 N 4 O 2 > 48.729 14 Lysine <![CDATA[C 6 H 14 N 2 O 2 > 54.098 15 Choline <![CDATA[C 5 H 15 NO 2 > 47.891

[0073] Example 3

[0074] In this example, the antioxidant effect of the Saussurea salwinensis cell culture extract was verified through an in vitro zebrafish oxidative stress model. The Saussurea salwinensis cell culture extract used in this example was obtained according to the extraction conditions of Experimental Example FMJ-14 in Example 1 (the ratio of material to liquid was 1:10, the concentration of edible ethanol was 70%, the number of extraction times was 3, and the single extraction time was 6 hours). Specifically, this example was carried out according to the following steps:

[0075] 1. Zebrafish embryo culture

[0076] Healthy zebrafish embryos were selected and cultured under the conditions of a water temperature of 28°C, a pH value of 7.0 - 7.5, and a conductivity of 400 - 600 μs / cm. They were fed three times a day, with 14 hours of fluorescent lamp irradiation and 10 hours of darkness.

[0077] 2. Grouping settings

[0078] Control group: Zebrafish embryos without any treatment;

[0079] Model group: 50 μM menadione was added to induce oxidative stress in zebrafish embryos;

[0080] Positive drug group: 50 μM menadione was added to induce oxidative stress in zebrafish embryos, and 50 μg / ml N-acetyl-L-cysteine was added as a positive control;

[0081] Antioxidant group 1: 50 μM menadione was added to induce oxidative stress in zebrafish embryos, and 0.1% fluid extract directly obtained from wild Saussurea salwinensis plants was added by total mass;

[0082] Antioxidant group 2: 50 μM menadione was added to induce oxidative stress in zebrafish embryos, and 0.5% fluid extract directly obtained from wild Saussurea salwinensis plants was added by total mass;

[0083] Antioxidant group 3: Add 50 μM menadione to induce oxidative stress in zebrafish embryos, and add the fluid extract of the cell culture extract induced by 0.1% Saussurea salwinensis Hand.-Mazz. callus by total mass;

[0084] Antioxidant group 4: Add 50 μM menadione to induce oxidative stress in zebrafish embryos, and add the fluid extract of the cell culture extract induced by 0.5% Saussurea salwinensis Hand.-Mazz. callus by total mass;

[0085] In all groups, add the corresponding treatment substances and continue culturing for 24 hours.

[0086] In this step, the treatment method of wild Saussurea salwinensis Hand.-Mazz. plants is the same as that of Saussurea salwinensis Hand.-Mazz. cell cultures.

[0087] 3. Reactive oxygen species (ROS) detection

[0088] The generation of ROS in zebrafish embryos of each group was measured using the DCFH-DA method. The DCFH-DA probe was diluted with serum-free culture medium at a ratio of 1:1000 to make its final concentration 10 μM. After removing the cell culture medium, add an appropriate volume of diluted DCFH-DA. For one well of a six-well plate, add no less than 1 mL of diluted DCFH-DA. Incubate the zebrafish embryos in a 37 °C cell culture incubator for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Detect the fluorescence intensity using a fluorescence microplate reader. The optimal excitation wavelength is 500 nm (500 ± 15 nm), and the optimal emission wavelength is 525 nm (530 ± 20 nm).

[0089] 4. Antioxidant capacity analysis

[0090] Taking the ROS fluorescence intensity of the blank group as 100% (baseline level), the ROS fluorescence intensity of the model group is 177.67 ± 12.06% of the blank group, the ROS fluorescence intensity of the positive drug group is 146 ± 8% of the blank group, the ROS fluorescence intensity of antioxidant group 1 is 144.33 ± 10.79% of the blank group, the ROS fluorescence intensity of antioxidant group 2 is 129 ± 4.58% of the blank group, the ROS fluorescence intensity of antioxidant group 3 is 115.33 ± 7.51% of the blank group, and the ROS fluorescence intensity of antioxidant group 4 is 87.67 ± 9.07% of the blank group.

[0091] From the above results, it can be seen that the extracts obtained from Saussurea salwinensis Hand.-Mazz. cell cultures show stronger antioxidant capacity than the extracts directly obtained from wild Saussurea salwinensis Hand.-Mazz. plants at the corresponding concentrations (0.1 wt% and 0.5 wt%).

[0092] Example 4

[0093] In this example, human skin fibroblasts (HSF) were used to verify the efficacy of the extract of Saussurea salwinensis cell culture in promoting collagen expression. The extract of Saussurea salwinensis cell culture used in this example was obtained according to the extraction conditions of Experimental Example FMJ-14 in Example 1 (the ratio of material to liquid was 1:10, the concentration of edible ethanol was 70%, the number of extraction times was 3 times, and the single extraction time was 6 hours). Specifically, this example was carried out according to the following steps:

[0094] 1. Culture of human skin fibroblasts (HSF)

[0095] The immortalized human skin fibroblasts were inoculated into a 6-well plate, with 2×10^5 cells per well, and 2 mL of medium containing 10% fetal bovine serum was added;

[0096] The above cells were cultured in a cell culture incubator at 37°C and 5% CO 2 until the cells reached 80-90% confluence.

[0097] 2. Setting of treatment groups

[0098] Blank control group: 2 mL of medium containing 10% fetal bovine serum was added;

[0099] Experimental group 1: 2 mL of medium containing 10% fetal bovine serum was added, and it contained 0.1% fluid extract prepared directly from wild Saussurea salwinensis plants;

[0100] Experimental group 2: 2 mL of medium containing 10% fetal bovine serum was added, and it contained 0.5% fluid extract prepared directly from wild Saussurea salwinensis plants;

[0101] Experimental group 3: 2 mL of medium containing 10% fetal bovine serum was added, and it contained 0.1% fluid extract prepared from Saussurea salwinensis cell culture;

[0102] Experimental group 4: 2 mL of medium containing 10% fetal bovine serum was added, and it contained 0.5% fluid extract prepared from Saussurea salwinensis cell culture.

[0103] In this step, the treatment method of wild Saussurea salwinensis plants was the same as that of Saussurea salwinensis cell culture.

[0104] 3. Pour out the medium in step 1, and then according to the treatment group settings in step 2, add new medium and continue to culture for 24 hours.

[0105] 4. Collect the cells, extract the total cellular RNA using TRIzol reagent, measure the RNA concentration and purity using a NanoDrop 2000 spectrophotometer (the A260 / A280 ratio should be between 1.8 and 2.0), and reverse transcribe 1 μg of total RNA into cDNA using SuperScript II reverse transcriptase with oligo dT (18 bp) as the primer. The reaction conditions are: 25 °C for 10 minutes; 42 °C for 50 minutes; 85 °C for 5 minutes.

[0106] 5. Use ChamQ Universal SYBR qPCR Master Mix from Nanjing Novoprotein as the reaction reagent, and detect the expression of human COL1A1 and COL3A1 genes in each treatment group using a CFX96 TOUCH real-time PCR instrument from Bio-Rad, with GAPDH as the internal reference gene. The qPCR primer sequences for each gene are shown in Table 3.

[0107] Table 3

[0108]

[0109] Figure 5 Shows the expression levels of COL1A1 and COL3A1 genes in human skin fibroblasts under different treatment conditions.

[0110] According to the qPCR detection results, the expression of COL1A1 and COL3A1 genes in HSF in experimental group 1 was not significantly affected; the expression of COL1A1 in HSF in experimental group 2 was increased by 2-fold compared with the control group, and the expression of COL3A1 was increased by 3.05-fold compared with the control group; the expression of COL1A1 in HSF in experimental group 3 was increased by 2.3-fold compared with the control group, and the expression of COL3A1 was increased by 2.98-fold compared with the control group; the expression of COL1A1 in HSF in experimental group 4 was increased by 4.5-fold compared with the control group, and the expression of COL3A1 was increased by 5.7-fold compared with the control group.

[0111] 7. Use the Yeasen Human Pro-Collagen I alpha 1 ELISA (Enzyme-Linked Immunosorbent Assay) kit to measure the content of type I collagen in HSF cells in different treatment groups. The specific steps are as follows:

[0112] Using the double antibody sandwich method, specific anti-human type I collagen antibodies are pre-coated on an enzyme-linked immunosorbent assay (ELISA) plate with high affinity. Standards and test samples are added to the wells of the ELISA plate. After incubation, the human type I collagen present in the samples binds to the solid-phase antibodies. After washing to remove unbound substances, a detection antibody (biotin-labeled) is added for incubation and binding. After washing again, an enzyme conjugate (HRP-labeled streptavidin) is added for incubation and binding. After washing, a chromogenic substrate TMB is added and the reaction is developed in the dark. The intensity of the color reaction is proportional to the concentration of human type I collagen in the sample. A stop solution is added to terminate the reaction, and the absorbance value is measured at a wavelength of 450 nm (reference wavelength 570 - 630 nm).

[0113] Figure 6 The results of the determination of the content of type I collagen in human skin fibroblasts under different treatment conditions are shown. There is no significant difference in the content of type I collagen in the cells of experimental group 1 compared with the blank control group; the content of type I collagen in the cells of experimental group 2 is increased by 1.36 times compared with the blank control group; the content of type I collagen in the cells of experimental group 3 is increased by 1.39 times compared with the blank control group; the content of type I collagen in the cells of experimental group 4 is increased by 2.49 times compared with the blank control group.

[0114] Therefore, the fluid extract of the extract directly obtained from Saussurea kingii plants at a concentration of 0.5% and the fluid extracts of the Saussurea kingii cell culture extracts at concentrations of 0.1% and 0.5% all have the effect of increasing the content of collagen in human fibroblasts. Among them, in terms of efficacy, the fluid extract of the extract directly obtained from Saussurea kingii plants at a concentration of 0.1% < the fluid extract of the extract directly obtained from Saussurea kingii plants at a concentration of 0.5% < the fluid extract of the Saussurea kingii cell culture extract at a concentration of 0.1% < the fluid extract of the Saussurea kingii cell culture extract at a concentration of 0.5%.

[0115] The solution of the present disclosure extracts active substances from Saussurea kingii cell cultures, which can avoid the influence of external factors such as raw material planting conditions, extraction parts, and seasons on the activity of the extracts, ensure the consistency and effectiveness of the extracts, and provide a reliable basis for the research on Saussurea kingii extracts in promoting collagen synthesis and antioxidant aspects.

[0116] Using Saussurea kingii cell cultures as raw materials and combining the extraction methods and parameters used in the present disclosure, extracts with higher total flavonoid content and richer other active ingredients can be obtained, showing more excellent antioxidant ability and collagen synthesis promotion ability compared with the extracts directly obtained from Saussurea kingii plants, providing a stable basis for the application of Saussurea kingii active substances in the pharmaceutical and cosmetic industries.

[0117] The above-described embodiments and application examples are only exemplary descriptions of the present disclosure, and do not limit the scope of the present disclosure. Without departing from the design spirit of the present disclosure, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present disclosure shall fall within the protection scope determined by the present disclosure.

Claims

1. A method for preparing a cell culture extract of Saussurea nujiangensis, characterized in that: The following steps are involved: S1. Collecting the cell culture of Saussurea nigra and drying it; S2. extracting the material obtained in step S1 with ethanol, wherein the volume ratio of the material to the ethanol is 1:5 to 1:20, the extraction time is 4 to 8 hours, and the volume fraction of the ethanol is 60 to 80%; S3. separating the extract and the extract residue in step S1, and centrifuging the extract to obtain a centrifuge; S4. Concentrate the centrifuge liquid to obtain a fluid extract.

2. The preparation method according to claim 1, characterized in that: In step S1, the cell culture of Saussurea nutans is dried to a water content of ≤10%.

3. The preparation method according to claim 1, characterized in that: In step S2, the volume ratio of the material to the ethanol is 1:10, the volume fraction of the ethanol is 70%, the single extraction time is 6 hours, and the extraction is performed 3 times.

4. The preparation method according to claim 1, characterized in that: In step S3, the extract and the extraction residue are separated by a sieve and a pipeline filter in sequence.

5. The preparation method according to claim 1, characterized in that: In step S3, the extract is concentrated before the centrifugal treatment, and the extract is concentrated to 20-30% of the original volume.

6. A cell culture extract of Saussurea nujiangensis, characterized in that: The extract is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the extract according to claim 6 in skin anti-oxidation.

8. Use of the extract according to claim 6 in promoting skin collagen synthesis.

9. A skin care composition, characterized in that: The composition comprises the extract of the cell culture of Saussurea niger according to claim 6.

10. The skin care composition according to claim 9, characterized in that: The added amount of the extract of the cell culture of Saussurea nutjiangense is 0.1% to 0.5% based on the total weight.

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