A saussurea involucrata callus extract, a preparation method and application thereof

Abstract

This invention belongs to the field of cosmetic technology and discloses an extract of *Saussurea involucrata* callus tissue, its preparation method, and its application. The preparation steps of the *Saussurea involucrata* callus tissue extract include: inoculating *Saussurea involucrata* in an induction culture medium to obtain *Saussurea involucrata* callus tissue; inoculating the *Saussurea involucrata* callus tissue in a growth culture medium, centrifuging to obtain *Saussurea involucrata* suspension cells; mixing the *Saussurea involucrata* suspension cells with an extraction solvent, homogenizing under high pressure to obtain the *Saussurea involucrata* callus tissue extract. This invention combines callus tissue culture, high-pressure homogenization, and a eutectic solvent, improving the efficiency of obtaining active ingredients from *Saussurea involucrata* callus tissue through a green process, and preparing a *Saussurea involucrata* callus tissue extract with significant anti-inflammatory effects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics and plant active ingredient extraction technology, specifically relating to a Tian Shan snow lotus callus extract, its preparation method, and its application. Background Technology

[0002] Tianshan snow lotus ( Saussurea involucrate *Saussurea involucrata* (Kar. et Kir.) is a rare traditional medicinal plant belonging to the genus *Saussurea* in the Asteraceae family, growing in the high-altitude and cold regions of China. It is commonly found in rock crevices, cliffs, and snowfields at altitudes of 4000–4500 meters. Due to its slow natural growth, unique growing environment, over-harvesting of wild resources, and increasingly deteriorating natural ecological environment, *Saussurea involucrata* has become an endangered species. Therefore, applying plant callus culture technology to the propagation of *Saussurea involucrata* and the production of its bioactive compounds can better protect *Saussurea involucrata* resources and effectively develop and utilize them, achieving the reusability of *Saussurea involucrata* plant resources. The active ingredients in *Saussurea involucrata* include flavonoids, anthocyanins, and phenylpropanoids. These components provide various effects, such as antioxidant, anti-inflammatory, anti-aging, and anti-UV damage effects. In the 2025 edition of the *Chinese Pharmacopoeia*, the identified components of *Saussurea involucrata* are rutin and chlorogenic acid, belonging to the flavonoid and phenylpropanoid classes, respectively. Flavonoids have various biological activities, such as geraniol, geraniol-7-O-glucoside, senna glycoside, sennaol and apigenin. However, these components are secondary metabolites with low synthesis efficiency and multiple transformation and metabolic pathways, making it difficult to efficiently enrich them in Tian Shan snow lotus and fully utilize the efficacy of Tian Shan snow lotus as a cosmetic ingredient.

[0003] Traditional methods for extracting flavonoids mainly rely on organic solvents such as ethanol, methanol, and ethyl acetate. While these methods are effective to some extent, they have some problems, such as solvent evaporation, environmental pollution, high cost, and limited extraction efficiency. Eutectic solvents are mixtures composed of hydrogen bond acceptors and hydrogen bond donors that form a homogeneous and transparent liquid under certain conditions. They have advantages such as diverse structures, low price, readily available raw materials, simple preparation, and no use of volatile organic solvents. In recent years, eutectic solvents have received increasing attention in the field of natural product extraction, especially in the extraction of flavonoids. Compared with traditional organic solvent extraction, eutectic solvents show the following advantages in the extraction of flavonoids: (1) High-efficiency extraction: Eutectic solvents can form hydrogen bonds and other interactions with flavonoids, thereby improving extraction efficiency. Some studies have shown that the use of eutectic solvents can significantly improve the extraction rate of flavonoids. (2) Low cost: Eutectic solvents have readily available raw materials, are simple to prepare, and have relatively low cost. This helps to reduce the cost of the extraction process and improve economic efficiency. (3) Green and environmentally friendly: The eutectic solvent does not use volatile organic solvents, which reduces environmental pollution and the risk of solvent volatilization. This is in line with the current concept of green chemistry and sustainable development. Summary of the Invention

[0004] The present invention aims to at least solve the technical problems existing in the prior art, realize the culture technology for preparing specific flavonoid components from Tian Shan snow lotus callus, and the green and efficient extraction of the flavonoid components.

[0005] Therefore, a first aspect of the present invention provides a method for preparing a callus extract from *Saussurea involucrata*, comprising the following steps:

[0006] S1. Tian Shan snow lotus was inoculated into an induction medium and cultured to obtain snow lotus callus tissue;

[0007] S2. The snow lotus callus tissue was inoculated into a growth medium and cultured, then centrifuged to obtain snow lotus suspension cells;

[0008] S3. Mix the snow lotus suspension cells and extraction solvent, and homogenize under high pressure to obtain the Tian Shan snow lotus callus extract.

[0009] Preferably, in step S1, the induction medium is based on MS medium and contains 30 g / L sucrose, 0.5-1.0 mg / L 6-benzylaminopurine, and 1.0-1.25 mg / L naphthaleneacetic acid. More preferably, the induction medium is based on MS medium and contains 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine, and 1.25 mg / L naphthaleneacetic acid. Even more preferably, the induction medium is based on MS medium and contains 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine, and 1.0 mg / L naphthaleneacetic acid.

[0010] Preferably, in step S1, the culture temperature of the snow lotus callus is 15-30℃, and the culture time is 30-40 days. More preferably, the culture temperature is 28℃, and the culture time is 35 days. Even more preferably, the culture medium is replaced every 7 days, and the culture medium is replaced 4-6 times during the culture period.

[0011] Preferably, in step S2, the growth medium is based on MS medium and contains 0.5-1.0 mg / L 6-benzylaminopurine, 1.0-1.25 mg / L naphthaleneacetic acid, and 30 g / L sucrose. More preferably, the growth medium is based on MS medium and contains 1.0 mg / L 6-benzylaminopurine, 12.5 mg / L naphthaleneacetic acid, and 30 g / L sucrose. Even more preferably, the growth medium is based on MS medium and contains 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine, and 1.0 mg / L naphthaleneacetic acid.

[0012] Preferably, in step S2, the culture temperature of the snow lotus suspension cells is 15-30℃, and the culture time is 20-35 days. More preferably, the culture temperature is 25℃, and the culture time is 28 days. Even more preferably, the culture medium is replaced every 7 days, and the culture medium is replaced 3-5 times during the culture period.

[0013] Preferably, in step S3, the mass ratio of the snow lotus suspension cells to the extraction solvent is 1:(6.5-20), more preferably 1:15.

[0014] Preferably, the extraction solvent is betaine, polyol, and water, and the mass ratio of betaine, polyol, and water is 1:(5-18):(0.5-4), most preferably 1:10:4. Preferably, the polyol is one or more of propylene glycol, butylene glycol, and glycerol.

[0015] Preferably, in step S3, the pressure of the high-pressure homogenization is 80-150 MPa, the temperature is 25-50°C, and the time is 5-30 min. More preferably, the pressure of the high-pressure homogenization is 130 MPa, the temperature is 40°C, and the time is 10 min.

[0016] Preferably, the Tian Shan snow lotus callus extract contains the following flavonoid components: total flavonoids 5.83-6.51 mg / g, geraniol 56.3-68.7 μg / g, geraniol-7-O-glucoside 6.47-8.87 μg / g, senna glycoside 0.83-1.13 μg / g, sennaol 0.02-0.03 μg / g, and apigenin 0.07-5.57 μg / g.

[0017] A second aspect of the present invention provides a callus extract from Tian Shan snow lotus, which is prepared by the above method.

[0018] A third aspect of the present invention provides the application of the above-mentioned Tian Shan snow lotus callus extract in the preparation of cosmetics.

[0019] A fourth aspect of the present invention provides a cosmetic product comprising the above-mentioned *Saussurea involucrata* callus extract and cosmetic-acceptable excipients, wherein the *Saussurea involucrata* callus extract accounts for 0.01-50% of the cosmetic product by mass.

[0020] The snow lotus callus extract provided by this invention has a high content of total flavonoids, as well as geraniol, geraniol-7-O-glucoside, senna glycoside, sennaol, and apigenin, thus exhibiting significant antioxidant and anti-inflammatory effects, and is mild and non-irritating. Detailed Implementation

[0021] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention. Rather, the purpose of providing these embodiments is to make the disclosure of this invention more thorough and comprehensive.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0024] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0025] In the following text, MS medium refers to the MS basal medium routinely used in plant tissue culture.

[0026] Example 1

[0027] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0028] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0029] 3) Dissolve 10g betaine and 100g propylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 40℃ and 130MPa for 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0030] Example 2

[0031] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine and 1.0 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) for callus induction. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0032] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 1.0mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0033] 3) Dissolve 10g betaine and 100g propylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 50℃ and 150MPa for 5min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0034] Example 3

[0035] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0036] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 1.0mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0037] 3) Dissolve 10g betaine and 130g propylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 10g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 25℃ and 80MPa for 30min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0038] Example 4

[0039] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0040] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 1.0mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0041] 3) Dissolve 10g betaine and 100g butylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 40℃ and 130MPa for 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0042] Example 5

[0043] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces.2 Leaf discs were inoculated into induction medium (MS medium based on 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine and 1.0 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) for callus induction. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0044] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 1.0mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0045] 3) Dissolve 10g betaine and 100g butylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 50℃ and 150MPa for 5min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0046] Example 6

[0047] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0048] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 1.0mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0049] 3) Dissolve 10g betaine and 50g butylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 5g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 25℃ and 80MPa for 30min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0050] Example 7

[0051] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0052] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0053] 3) Dissolve 10g betaine and 100g glycerol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and add to a high-pressure homogenizer for high-pressure homogenization at 40℃ and 130MPa for 10min. After filtration, the Tian Shan snow lotus callus extract is obtained.

[0054] Example 8

[0055] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine and 1.0 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) for callus induction. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0056] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 1.0mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0057] 3) Dissolve 10g betaine and 100g glycerol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 50℃ and 150MPa for 30min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0058] Example 9

[0059] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0060] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0061] 3) Dissolve 10g betaine and 180g glycerol at 60℃, then mix with 10g snow lotus suspension cells and 10g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 25℃ and 80MPa for 30min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0062] Comparative Example 1

[0063] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on 30 g / L sucrose, 3.0 mg / L 6-benzylaminopurine and 0.5 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) for callus induction. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0064] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 3.0mg / L 6-benzylaminopurine, and 0.5mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0065] 3) Dissolve 10g betaine and 100g propylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 40℃ and 130MPa for 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0066] Comparative Example 2

[0067] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2Leaf discs were inoculated into induction medium (MS medium based on MS medium supplemented with 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L dichlorophenoxyacetic acid, and solidified with 7 g / L agar) for callus induction. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0068] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L 2,4-dichlorophenoxyacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0069] 3) Dissolve 10g betaine and 100g propylene glycol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 40℃ and 130MPa for 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0070] Comparative Example 3

[0071] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0072] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0073] 3) Dissolve 10g betaine and 100g 1,2-hexanediol at 60℃, then mix with 10g snow lotus suspension cells and 40g water, and then add to a high-pressure homogenizer for high-pressure homogenization at 40℃ and 130MPa for 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0074] Comparative Example 4

[0075] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0076] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0077] 3) Mix 10g of snow lotus suspension cells, 110g of ethanol and 40g of water, and then add them to a high-pressure homogenizer for high-pressure homogenization. The homogenization temperature is 40℃, the homogenization pressure is 130MPa, and the homogenization time is 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0078] Comparative Example 5

[0079] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2 Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0080] 2) Take 5g of snow lotus callus tissue, add growth medium (based on MS medium, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After centrifuging the culture medium at 5000rpm for 15min, discard the supernatant to obtain snow lotus suspension cells.

[0081] 3) Mix 10g of snow lotus suspension cells, 110g of propylene glycol and 40g of water, and then add them to a high-pressure homogenizer for high-pressure homogenization. The homogenization temperature is 40℃, the homogenization pressure is 130MPa, and the homogenization time is 10min. After filtration of the homogenized liquid, the Tian Shan snow lotus callus extract is obtained.

[0082] Comparative Example 6

[0083] 1) Take leaves from the Tian Shan snow lotus, cut off the leaf edges to make 1 cm pieces. 2Leaf discs were inoculated into induction medium (MS medium based on MS medium with the addition of 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine and 1.25 mg / L naphthaleneacetic acid, and solidified with 7 g / L agar) to induce callus formation. The medium was changed every 7 days, and after culturing at 28°C for 35 days, snow lotus callus was obtained.

[0084] 2) Take 5g of snow lotus callus tissue, add it to the growth medium (MS medium as the base, with 30g / L sucrose, 1.0mg / L 6-benzylaminopurine, and 1.25mg / L naphthaleneacetic acid added), and culture at 25℃ for 28 days. After the culture medium is centrifuged at 5000rpm for 15min, the supernatant is discarded to obtain snow lotus suspension cells.

[0085] 3) Mix 10g of snow lotus suspension cells, 110g of ethanol and 40g of water, reflux extract at 80℃ for 1h, filter, and obtain the Tian Shan snow lotus callus tissue heated reflux extract.

[0086] Comparative Example 7

[0087] 10g of betaine and 100g of propylene glycol were mixed and dissolved at 60℃. Then, the mixture was added to 10g of snow lotus leaf powder and 40g of water and homogenized in a high-pressure homogenizer at 40℃ and 130MPa for 10 minutes. After filtration, the snow lotus leaf extract was obtained.

[0088] Experimental Example 1: Determination of Total Flavonoid Content

[0089] This experiment tested the total flavonoid content in the callus extracts of Tian Shan snow lotus from Examples 1-9 and the extracts of Tian Shan snow lotus from Comparative Examples 1-7 (hereinafter referred to as "samples").

[0090] The specific testing method is as follows:

[0091] Adjust the total flavonoid concentration of the sample solution to the determination range, accurately pipette 2.0 mL into a dry, clean test tube, add 0.2 mL of 5% (w / w) sodium nitrite solution, shake well and let stand for 5 min, then add 0.2 mL of 10% (w / w) aluminum nitrate solution and shake well. Let stand for 6 min, then add 1.0 mL of 5% (w / w) sodium hydroxide solution, and add solvent to the mark. Shake well the sample solution. Quickly measure the absorbance of each tube at a wavelength of 510 nm. Calculate the total flavonoid concentration of the sample solution from the absorbance value based on the standard curve of rutin solution. The total flavonoid content in the sample is expressed as rutin.

[0092] Experimental Example 2: Determination of the contents of geraniol, geraniol-7-O-glucoside, buddleja glycoside, sennaol, and apigenin.

[0093] This test example examines the flavonoid content in the callus extracts of *Saussurea involucrata* from Examples 1-9 and the extracts of *Saussurea involucrata* from Comparative Examples 1-7 (hereinafter referred to as "samples").

[0094] The specific testing method is as follows:

[0095] Sample preparation: Take 1.0 g of sample and place it in a 10 mL volumetric flask. Add chromatographic grade methanol to the mark, shake well, and filter through a 0.22 μm filter membrane to obtain the test sample.

[0096] LC-MS / MS analysis conditions:

[0097] Column: Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 µm);

[0098] Mobile phase: Phase A is ultrapure water (containing 0.1% v / v formic acid), and Phase B is acetonitrile (containing 0.1% v / v formic acid).

[0099] Elution gradient: 0 min 5% phase B, 1 min 22% phase B, 6 min 22% phase B, 8 min 40% phase B, 11 min 95% phase B, 12 min 95% phase B;

[0100] Flow rate 0.40 mL / min, column temperature 38 °C, injection volume 3 µL;

[0101] Mass spectrometry parameters: electrospray ionization source temperature 550 °C; ionization source voltage 5500 V; curtain gas 35 psi; collision-induced ionization 2.

[0102] The test results for Experiment 1 and Experiment 2 are shown in Table 1.

[0103] Table 1

[0104]

[0105] ND indicates not detected.

[0106] The samples in Examples 1-9 contained the following flavonoids: total flavonoids 5.83-6.51 mg / g, geraniol 56.3-68.7 μg / g, geraniol-7-O-glucoside 6.47-8.87 μg / g, senna glycoside 0.83-1.13 μg / g, sennaol 0.02-0.03 μg / g, and apigenin 0.07-5.57 μg / g.

[0107] As shown in Table 1, compared with Comparative Examples 1, 2, 3, 5, and 7, the total flavonoid content, geraniol, geraniol-7-O-glucoside, sennain, sennaol, and apigenin content in the samples of Examples 1-9 were significantly increased. This indicates that the culture process of *Saussurea involucrata* callus can significantly improve the generation of specific flavonoid components. The betaine and diols or triols used in the extraction process can form a eutectic solvent, which can efficiently penetrate *Saussurea involucrata* callus and has a more advantageous interaction with flavonoid components, making the components easier to dissolve. Compared with Comparative Examples 4 and 6, the total flavonoid content and the content of each compound were also increased, indicating that the use of organic solvents and the risk of solvent residue can be avoided when efficiently obtaining flavonoid components from *Saussurea involucrata* callus, achieving green and efficient active ingredient extraction. In the induction and growth media of Examples 2, 5, and 8, the concentration ratio of 6-benzylaminopurine to naphthaleneacetic acid was 1:2. At this ratio, the content of apigenin is high, but the content of monaxioline is low.

[0108] Experimental Example 3: DPPH Free Radical Scavenging Ability

[0109] This experiment tested the DPPH free radical scavenging ability of the Tian Shan snow lotus callus extracts of Examples 1-9 and the Tian Shan snow lotus extracts of Comparative Examples 1-7 (hereinafter referred to as "samples").

[0110] The specific testing method is as follows:

[0111] Add 2.0 mL of DPPH test solution with a mass concentration of 45.8 mg / L and 0.5 mL of the sample to be tested to a test tube. The total volume is then increased to 3 mL with 50% ethanol (v / v). Shake well and react in the dark for 30 min. Measure the absorbance at 517 nm using a 1 cm cuvette and record it as A1. Add 2.0 mL of anhydrous ethanol and the corresponding volume of the sample to be tested to a test tube. The total volume is then increased to 3.0 mL with 50% ethanol (v / v). Record the measured absorbance as A2. Add 2.0 mL of DPPH test solution and 1 mL of 50% ethanol (v / v) to a test tube. Record the measured absorbance as A3.

[0112] The calculation formula is as follows: DPPH radical scavenging rate = (1 - (A3 - A2) / A1) × 100%. IC is calculated using R software. 50 value.

[0113] Experimental Example 4: OH Free Radical Scavenging Ability

[0114] This experiment tested the OH radical scavenging ability of the Tian Shan snow lotus callus extracts of Examples 1-9 and the Tian Shan snow lotus extracts of Comparative Examples 1-7 (hereinafter referred to as "samples").

[0115] The specific testing method is as follows:

[0116] The reaction system was first supplemented with 1.0 mL FeSO4 solution (9.0 mmol / L), 1 mL salicylic acid-ethanol solution (9.0 mmol / mL), and 1.0 mL sample solution, followed by 1.0 mL H2O2 (8.8 mmol / mL). The reaction was carried out in a water bath at 37 °C for 30 min. Anhydrous ethanol was used as a blank control, and the absorbance was measured at 510 nm.

[0117] The calculation formula is as follows: OH radical scavenging rate = [1 - (A1 - A2) / A3] × 100%, where A1 is the absorbance of the extraction solution reaction system; A2 is the absorbance of the sample without H2O2 (using anhydrous ethanol instead of H2O2); and A3 is the absorbance of the blank control anhydrous ethanol. IC50 was calculated using R software. 50 value.

[0118] Experimental Example 5: Determination of Total Antioxidant Capacity by FRAP Method

[0119] This experiment tested the total antioxidant capacity of the Tian Shan snow lotus callus extracts from Examples 1-9 and the Tian Shan snow lotus extracts from Comparative Examples 1-7 (hereinafter referred to as "samples").

[0120] The specific testing method is as follows:

[0121] (1) Sample determination: Add 100 μL of sample solution (dilute appropriately as needed) to the reaction tube, then add 3 mL of FRAP working solution, mix well, react at 37 °C for 15 min, and measure the absorbance at 593 nm.

[0122] (2) Determination of FeSO4 standard curve: According to the sample determination procedure, a standard curve was plotted using a standard solution of FeSO4 with a concentration of 0.1-1.6 mmol / L instead of the sample. The final total antioxidant activity of the sample was expressed as the equivalent concentration of FeSO4 in mmol / L. Using FeSO4 as the standard solution, the concentration of FeSO4 (mmol / L) was calculated on the standard curve based on the A value after the reaction, and defined as the FRAP value. The higher the FRAP value, the stronger the antioxidant activity.

[0123] The test results for Experiments 3, 4 and 5 are shown in Table 2.

[0124] Table 2

[0125]

[0126] As shown in Table 2, compared with Comparative Examples 1-7, the DPPH free radical scavenging rate IC50 of the Tian Shan snow lotus callus extracts in Examples 1-9 was significantly higher. 50IC50 value and OH radical scavenging rate 50 The values ​​were all low, indicating a stronger ability to scavenge free radicals. Furthermore, the Tian Shan snow lotus callus extracts from Examples 1-9 showed a stronger effect in terms of total antioxidant capacity.

[0127] Based on the results of three antioxidant experiments, the Tian Shan snow lotus callus extracts in Examples 1-9 contained more potential antioxidant components.

[0128] Experimental Example 6: Evaluation of Anti-inflammatory Activity

[0129] This study evaluated the anti-inflammatory activity of the extract on the production of nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7. Mouse macrophages RAW264.7 were cultured in DMEM complete medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-drug antibiotics) at 37°C in a 5% CO2 incubator, passaged every 2–3 days. RAW264.7 cells in logarithmic growth phase were cultured at a rate of 1 × 10⁻⁶ cells / cell. 5 / wells were seeded in 96-well plates with 200 μL of DMEM complete medium per well. After 24 h of culture, 1 μg / mL LPS and 4% (v / v) of each sample were added. The blank group was added with 4% (v / v) DMEM complete medium, and the model group was added with 1 μg / mL LPS and 4% (v / v) DMEM complete medium. After culturing for another 24 h, the supernatant was collected, and the production of NO, COX-2, TNF-α, and IL-6 was determined according to the kit instructions.

[0130] The test results are shown in Table 3.

[0131] Table 3

[0132]

[0133] As shown in Table 3, compared with Comparative Examples 1-7, the snow lotus callus extracts of Examples 1-9 can more effectively reduce the production of NO, COX-2, TNF-α and IL-6, exhibiting a stronger anti-inflammatory effect.

[0134] Test Example 7: Eye Irritation / Corrosiveness of Chicken Embryo Villi Allantoic Membrane Experiment

[0135] Nine-day-old chicken embryos weighing 50–60g were selected. The air cell was marked on the eggshell surface. The eggshell was partially removed with tweezers to expose the white egg membrane. 0.9% w / w physiological saline was added with a pipette to moisten the egg membrane. The inner membrane was removed with tweezers. 0.3 mL of the sample was applied directly to the chorioallantoic membrane (CAM), ensuring that the coverage area was at least 50%. The CAM reaction was observed immediately and stopped after 5 minutes. The observation results were recorded and judged. Each sample was repeated 3 times. Physiological saline was used as a negative control. IS < 1 indicates no irritation, 1 ≤ IS < 5 indicates mild irritation, and 5 ≤ IS < 10 indicates moderate irritation.

[0136] The test results are shown in Table 4.

[0137] Table 4

[0138]

[0139] Table 4 shows that the snow lotus callus extracts from Examples 1-9 and Comparative Examples 1, 2, 3, 5, and 7 were non-irritating, while the snow lotus callus extracts from Comparative Examples 4 and 6 were mildly irritating. The results indicate that the snow lotus callus extracts prepared in Examples 1-9 are relatively mild raw materials.

[0140] Application Example 1

[0141] This application example provides an emulsion containing the Tian Shan snow lotus callus extract from Example 1, the formulation of which is shown in Table 5 by mass percentage:

[0142] Table 5

[0143]

[0144] Application Comparative Example 1

[0145] The difference between Comparative Example 1 and Application Example 1 is that Comparative Example 1 replaces the Tian Shan snow lotus callus extract of Example 1 with the Tian Shan snow lotus leaf extract of Comparative Example 7. Other components, dosages and preparation methods are the same as in Application Example 1.

[0146] Twenty volunteers, aged 20-45 years, with an equal number of men and women, were selected and divided into two groups. One group used a lotion containing Application Example 1, and the other used a lotion containing Comparative Example 1. The test site was the face. After cleansing morning and evening, participants applied an appropriate amount of product evenly to the face and massaged until absorbed. The product was used twice daily, once in the morning and once in the evening. Follow-up was conducted before product use (Day 0), 14 days after product use, and 28 days after product use. Evaluation indicators included skin erythema and desquamation at the test site, with each indicator rated from 0 (very good) to 5 (very poor) based on its severity. Additionally, the repairing and soothing efficacy of the product was assessed by comparing skin hydration, transepidermal water loss (TEWL), and the area of ​​skin redness before and after product use.

[0147] The results are shown in Table 6.

[0148] Table 6

[0149]

[0150] As shown in Table 6, compared with the lotion of Comparative Example 1, the subjects showed improvement in skin erythema and desquamation after using the lotion of Application Example 1. Combined with the changes in stratum corneum moisture content, TEWL value and red area area before and after use, it indicates that the Tian Shan snow lotus callus extract of the present invention has significant soothing and repairing functions.

[0151] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for preparing an extract from callus tissue of *Saussurea involucrata*, characterized in that, Includes the following steps: S1. Tian Shan snow lotus was inoculated into an induction medium and cultured to obtain snow lotus callus tissue; S2. The snow lotus callus tissue was inoculated into a growth medium and cultured, then centrifuged to obtain snow lotus suspension cells; S3. Mix the snow lotus suspension cells and extraction solvent, homogenize under high pressure to obtain the Tian Shan snow lotus callus extract; In step S1, the induction medium is based on MS medium and contains 30 g / L sucrose, 0.5-1.0 mg / L 6-benzylaminopurine and 1.0-1.25 mg / L naphthaleneacetic acid; In step S2, the growth medium is based on MS medium and contains 30 g / L sucrose, 0.5-1.0 mg / L 6-benzylaminopurine and 1.0-1.25 mg / L naphthaleneacetic acid; In step S3, the mass ratio of the snow lotus suspension cells to the extraction solvent is 1:(6.5-20); The extraction solvent is betaine, polyol and water, and the mass ratio of betaine, polyol and water is 1:(5-18):(0.5-4). The polyol is one or more of propylene glycol, butylene glycol and glycerol. In step S3, the pressure of the high-pressure homogenization is 80-150 MPa, the temperature is 25-50℃, and the time is 5-30 min.

2. The method according to claim 1, characterized in that, In step S1, the culture temperature of the snow lotus callus is 15-30℃, and the culture time is 30-40 days.

3. The method according to claim 1 or 2, characterized in that, In step S2, the culture temperature of the snow lotus suspension cells is 15-30℃, and the culture time is 20-35 days.

4. An extract from the callus tissue of *Saussurea involucrata*, characterized in that, It is prepared by the method described in any one of claims 1 to 3.

5. The Tian Shan snow lotus callus extract according to claim 4, characterized in that, The Tian Shan snow lotus callus extract contains the following flavonoid components: total flavonoids 5.83-6.51 mg / g, geraniol 56.3-68.7 μg / g, geraniol-7-O-glucoside 6.47-8.87 μg / g, senna glycoside 0.83-1.13 μg / g, sennaol 0.02-0.03 μg / g, and apigenin 0.07-5.57 μg / g.

6. The application of the Tian Shan snow lotus callus extract as described in claim 4 in the preparation of cosmetics.

7. A cosmetic product, characterized in that, The product includes the Tian Shan Snow Lotus callus extract as described in claim 4 and cosmetic-acceptable excipients, wherein the Tian Shan Snow Lotus callus extract accounts for 0.01-50% of the cosmetic product by mass.

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