Saussurea lancea callus proliferation culture medium, saussurea lancea callus extract, preparation method and application

Abstract

The invention discloses a saussurea lancea callus proliferation culture medium, a saussurea lancea callus extract, a preparation method and application. The saussurea lancea callus proliferation culture medium comprises the following components: a basic culture medium for plant tissue culture and 3-100 ppm of nisin z. Compared with an existing saussurea lancea culture medium, the saussurea lancea callus proliferation culture medium can solve the technical problem that the saussurea lancea callus is prone to browning during large-scale production, and meanwhile the saussurea lancea callus proliferation coefficient can be increased, and the content of active ingredients in an extract can be increased; the saussurea lancea callus with high multiplication coefficient and the saussurea lancea callus extract with improved oxidation resistance are obtained.

Description

Technical Field

[0001] The present invention relates to a saussurea involucrata callus proliferation culture medium and extract, as well as a preparation method and application thereof Background Art

[0002] Snow lotus, with its high medicinal and ornamental value, is a key Tibetan medicinal plant resource. It is primarily found in the high-altitude regions of western my country and is less common abroad. Saussurea scabra (Saussurea scabra), also known as the cotton-headed snow rabbit, is a perennial herbaceous plant in the genus Saussurea (DC.) of the Asteraceae family (Compositae). It is a Class II National Protected Plant. Its flowering and fruiting period is from August to October, and its leaves and inflorescences are covered with thick, woolly hairs. It is found in Sichuan, Yunnan, and Tibet, primarily on alpine rocky plains at altitudes between 3,200 and 5,280 meters, making it a highly representative alpine plant in my country. Research has shown that Saussurea scabra is rich in flavonoids, polyphenols, polysaccharides, and secondary metabolites, exhibiting free radical scavenging, anti-aging, and analgesic properties. Saussurea scabra primarily reproduces through seeds, but intensive harvesting disrupts the ecological balance and severely affects its natural growth and reproduction. Therefore, using plant tissue culture to obtain callus cells from saussurea seeds is an effective way to preserve germplasm and address the endangered status of saussurea.

[0003] During the culture process of Saussurea chinensis callus, it was found that the callus proliferation rate of Saussurea chinensis cultured using ordinary culture medium was low at the beginning of culture, resulting in a lower yield of Saussurea chinensis callus, and the content of active ingredients such as flavonoids, polysaccharides, and polyphenols in the callus extract was low, limiting its further expansion of production and application. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defect in the prior art that the skin care efficacy of the cell extract obtained by culturing Saussurea scoparia callus tissue with a culture medium needs to be improved, thereby providing a Saussurea scoparia callus tissue proliferation culture medium and extract, preparation method and application. Compared with the existing culture medium, the Saussurea scoparia callus tissue proliferation culture medium provided by the present invention can solve the technical problem that Saussurea scoparia callus tissue is easy to brown during large-scale production, and at the same time can also improve the proliferation coefficient of Saussurea scoparia callus tissue and increase the content of active ingredients in the extract, thereby obtaining Saussurea scoparia callus tissue with a high proliferation coefficient, and Saussurea scoparia callus tissue extract containing total polyphenols, total flavonoids and polysaccharides, and having improved antioxidant capacity.

[0005] The present invention solves the above technical problems through the following solutions: A callus proliferation culture medium for Saussurea scoparia, characterized in that it comprises the following components: Basic medium for plant tissue culture, nisin 3-100 ppm; wherein: Ppm refers to the concentration in the Saussurea involucrata callus proliferation culture medium.

[0006] Nisin, also known as "nisin", is commonly used in dairy products (cheese, yogurt), canned food, and meat products to extend the shelf life of food.

[0007] Furthermore, the concentration of nisin z is 4 to 80 ppm.

[0008] Furthermore, the basal culture medium is selected from MS culture medium, N6 culture medium, B5 culture medium, SH culture medium or White culture medium, preferably MS culture medium; Furthermore, the Saussurea involucrata callus proliferation culture medium further comprises plant hormones; the plant hormones are preferably selected from one or more of NAA, 6-BA, 2,4-D and KT; more preferably, 6-BA and NAA; Furthermore, the concentration of the plant hormone in the Saussurea involucrata callus proliferation culture medium is preferably 0.1 to 2.0 mg / L, more preferably 0.2 to 1.8 mg / L; When the plant hormones are 6-BA and NAA, the concentration of 6-BA in the Saussurea scoparia callus proliferation culture medium is 0.3-0.7 mg / L, and the concentration of NAA in the Saussurea scoparia callus proliferation culture medium is preferably 0.3-0.7 mg / L; and / or, the Saussurea scoparia callus proliferation culture medium further comprises a carbon source, such as one or more of sucrose, maltose and glucose, preferably sucrose; and the concentration of the carbon source in the Saussurea scoparia callus proliferation culture medium is preferably 28-35 g / L, more preferably 30 g / L; And / or, the Saussurea scoparia callus proliferation culture medium further comprises a coagulant, such as agar and / or gellan gum, preferably gellan gum; the concentration of the coagulant in the Saussurea scoparia callus proliferation culture medium is preferably 3-4 g / L, more preferably 3.5 g / L; And / or, the Saussurea involucrata callus proliferation culture medium further comprises potato flour; the concentration of the potato flour is 1-35 g / L, preferably 30 g / L.

[0009] Preferably, the culture medium is MS+6-BA 0.5mg / L+NAA 0.5mg / L+nisin z 5ppm+sucrose 30g / L+gellan gum 3.5g / L+potato flour 30g / L or MS+6-BA 0.5mg / L+NAA 0.5mg / L+nisin z 50ppm+sucrose 30g / L+gellan gum 3.5g / L+potato flour 30g / L.

[0010] The present invention also provides a method for preparing a culture medium for increasing the callus tissue of Saussurea scoparia. The method comprises the following steps: mixing the components of the culture medium for increasing the callus tissue of Saussurea scoparia.

[0011] The present invention also provides a method for increasing the proliferation rate of Saussurea scoparia callus, wherein the Saussurea scoparia callus is cultured using the Saussurea scoparia callus proliferation culture medium described above.

[0012] The present invention also provides a method for preventing browning of Saussurea scoparia callus, wherein the Saussurea scoparia callus is cultured using the Saussurea scoparia callus proliferation culture medium described above.

[0013] The Saussurea involucrata callus as described above is induced by the following method: sterilizing seeds of wild Saussurea involucrata and placing them on MS basal medium in the dark for cultivation, and transferring the seeds to MS culture bottles for cultivation after germination and elongation to about 1 cm to obtain sterile seedlings; The roots, stems and leaves of the sterile seedlings were cut into segments of 0.5 cm to 1.0 cm in length and inoculated into an induction medium of MS + 0.5 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 3.5 g / L gellan gum. After dark culture, dense tissue appeared at the incision. After continuing to culture for 30 days, the cotton callus tissue was obtained.

[0014] The present invention also provides a Saussurea scoparia callus cell extract, which is characterized in that it is obtained by extracting cultured Saussurea scoparia callus cells, and the cultured Saussurea scoparia callus cells are obtained by culturing Saussurea scoparia callus tissue in the Saussurea scoparia callus proliferation culture medium described above.

[0015] The conditions for culturing the Saussurea scoparia callus are: temperature of 20±1°C, humidity of 50%-70%, and dark culturing; and the extraction method comprises the following steps: taking the Saussurea scoparia callus, crushing it according to a material-liquid ratio of 1:10-1:30, then extracting it at a temperature of 75-85°C for 2-4 hours, centrifuging it at 8000-12000 rpm for 40-80 minutes, filtering and obtaining the supernatant, and freeze-drying the supernatant into powder to obtain the Saussurea scoparia callus extract powder.

[0016] And / or, the polyphenol content in the extract of Saussurea involucrata callus cells is 2-2.2% And / or, the content of flavonoids in the extract of Saussurea scoparia callus cells is 3-3.5%. The content of polyphenols or flavonoids refers to the mass percentage of polyphenols or flavonoids in the extract of Saussurea scoparia callus cells.

[0017] The present invention also provides an application of an extract of callus cells of Saussurea scoparia in preparing a skin external preparation, characterized in that the extract of callus cells of Saussurea scoparia is used as the skin external preparation, an antioxidant active ingredient, an anti-aging active ingredient or a whitening active ingredient in a health food.

[0018] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0019] The reagents and raw materials used in the present invention are commercially available.

[0020] The positive progress effect of the present invention is: 1) The present invention's callus proliferation culture medium, by adding nisin Z, can address the technical issue of browning during large-scale production of Saussurea scoparia callus. Furthermore, when used to culture unbrown Saussurea scoparia callus, it can significantly increase the proliferation coefficient of Saussurea scoparia callus cells compared to existing culture media.

[0021] 2) The Saussurea scoparia callus cell extract obtained by culturing the Saussurea scoparia callus proliferation medium of the present invention contains higher total polyphenols, total flavonoids and polysaccharides, and has stronger antioxidant capacity.

[0022] 3) The Saussurea scabra callus obtained by the present invention has the advantages of stable subculture, short culture cycle, green environmental protection, stable quality, etc., which solves the problem that the wild Saussurea scabra plant resources are difficult to develop and utilize, and has good application prospects in the cosmetics, health care products and pharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effects of nisin z on the callus growth of Saussurea scabra (a) Blank group - no nisin added (b) Nisin group - 5PPM nisin z added DETAILED DESCRIPTION

[0024] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0025] Example 1 Callus Induction of Saussurea involucrata 1) Culture Medium Preparation and Culture Conditions Sterile seedlings of Saussurea chinensis callus tissue are cultured on MS basal medium. The induction medium is selected from one of MS, B5, or SH media, supplemented with various plant growth regulators, cytokinins, and other nutrients. Each medium is supplemented with 30g / L sucrose and 3.5g / L gellan gum, and the pH is adjusted to 5.7-5.8. The medium is then sterilized in an autoclave at 121°C for 20 minutes before use. Daily culture conditions are a temperature of 20±1°C, a humidity of 50%-70%, and dark incubation.

[0026] The basal medium MS (M519), B5 (G398), SH (816), PPM plant tissue culture antimicrobial agent, and plant growth regulators were all purchased from Pytotechnolagy. The plant growth regulators included naphthaleneacetic acid (NAA), 6-benzylaminoadenine (6-BA), and 2,4-dichlorophenoxyacetic acid (2,4-D). Nisin Z, CAS number 1414-45-5, was purchased from Hangzhou Enhe Biotechnology Co., Ltd.

[0027] 2). Seed germination and callus induction of Saussurea involucrata After pruning, the seeds of wild Saussurea involucrata were rinsed under running water for 20 minutes and then washed five times with sterile water. They were then sterilized in a sterile laminar flow hood with 75% ethanol (with Tween 20) for 15 seconds and rinsed five times with sterile water. The seeds were then shaken with a 2% (v / v) NaClO solution (with Tween 20) at 110 rpm for 10 minutes, rinsed five times with sterile water, and evenly plated on MS basal medium and cultured in the dark at 13°C. Once the seeds germinated and grew to approximately 1 cm, they were transferred to MS culture bottles for culture to obtain sterile seedlings.

[0028] In a sterile clean bench, the roots, stems, and leaves of sterile Saussurea involucrata seedlings were cut into 0.5-1.0 cm segments with a scalpel and inoculated into an induction medium consisting of MS + 0.5 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 3.5 g / L gellan gum. After dark culture for about two weeks, it was observed that the segments were significantly enlarged and dense tissue appeared at the incision. After another 30 days of culture, yellow-green callus tissue was visible.

[0029] 3). Screening of callus tissue (1) In order to screen out the callus tissue of Saussurea scabra in good condition, the callus tissue induced by the above steps was taken and inoculated into culture medium containing MS, B5, SH, and added with different proportions of 6-BA, 2,4-D or NAA for culture. Each culture medium was added with 30 g / L sucrose and 3.5 g / L gellan gum. The culture medium was prepared with deionized water. Four parallel groups were inoculated for each treatment, and the cell status was observed after 30 days of culture.

[0030] The results are shown in Table 1 Table 1. Effects of different culture medium formulas on the growth of Saussurea edulis callus

[0031] Different culture medium formulations yielded callus tissue in varying states. Visual inspection combined with sterile forceps gripping evaluated the callus's browning and texture. Culture mediums 1 and 2 produced calli that were yellow and relatively loose, indicating optimal callus conditions suitable for screening and culture. Cultures in other culture medium formulations tended to cause cell differentiation or softening, making it difficult to maintain callus cell status and survival. Therefore, based on the results in Table 1, the optimal culture medium formulation was MS + 0.5 mg / L 6-BA + 0.3-0.5 mg / L NAA + 30 g / L sucrose + 3.5 g / L gellan gum.

[0032] 4). Screening of culture medium for Saussurea scabra To further identify a culture medium suitable for the proliferation and culture of Saussurea scoparia calli, the yellow granular callus No. 1 from step 1 was selected and subjected to three further acclimation and culture cycles. Cells from these three acclimation cycles were inoculated into the following culture media with different formulations, each supplemented with 30 g / L sucrose and 3.5 g / L gellan gum. Four parallel groups were cultured for each treatment. After 30 days of culture, the cell status was observed and the callus cell proliferation coefficient was calculated.

[0033] Callus cell proliferation coefficient = cell weight 30 days after inoculation / cell weight on day 0 of inoculation. The results are shown in Table 2: Table 2. Effects of different culture medium formulas on the growth of Saussurea edulis callus

[0034] Adding potato flour to the culture medium resulted in noticeable callus expansion, yellow color, no differentiation or browning, and an increased proliferation coefficient. In summary, MS + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L potato flour + 30 g / L sucrose + 3.5 g / L gellan gum was the optimal culture medium formulation.

[0035] 5). Expanded scale culture of Saussurea involucrata callus To obtain more callus, the above-mentioned culture medium formula MS+6-BA 0.5mg / L+NAA 0.5mg / L+potato flour 30g / L+sucrose 30g / L+gellan gum 3.5g / L was used to expand the scale culture. However, after expanding the culture twice, the callus still showed browning and death during the culture process. Callus browning is a common problem in plant tissue culture, mainly caused by the oxidation of phenolic substances, resulting in cell death, growth inhibition and even culture failure. In the scale culture process, the callus of Saussurea scabra without browning was selected and the nisin z samples of different concentrations were added to test. Each culture medium formula was added with 30g / L sucrose and 3.5g / L gellan gum. 50 parallel groups were used for each treatment. Cell status was observed after 30 days of cultivation, and the browning rate and callus cell proliferation coefficient were statistically analyzed.

[0036] Table 3. Effects of different nisin z additions on callus growth of Saussurea scabra

[0037] The results showed that when the No. 1 medium without acid streptozocin Z was used for scale-up production, the callus of Saussurea scabra was easily browned. Figure 1 Compared to medium No. 1, the browning rate of callus cultured in medium supplemented with nisin Z was significantly reduced, and this rate was concentration-dependent within a certain concentration range. Furthermore, in the experimental groups containing 1-50 ppm of nisin Z, calli grew normally. The 5-50 ppm group showed slight expansion and an increasing proliferation coefficient. However, in the 75 ppm, 500 ppm, and 5000 ppm groups, calli showed a decrease in proliferation rate and even death as the nisin Z concentration increased. In summary, the optimal proliferation medium formulation was selected: MS + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L potato flour + 5-50 ppm L-nisin + 30 g / L sucrose + 3.5 g / L gellan gum.

[0038] However, the use of the conventional plant tissue culture antimicrobial agent PPM No. 8 culture medium cannot solve the technical problem of browning of the Saussurea chinensis callus tissue.

[0039] To further explore the effect of potato flour addition on callus growth, the following experiment was designed. Each culture medium consisted of a base formulation consisting of MS, 0.5 mg / L 6-BA, 0.5 mg / L NAA, and 5 ppm nisin, supplemented with 30 g / L sucrose and 3.5 g / L gellan gum. Fifty replicates were used for each treatment. After 30 days of culture, cell status was observed, and the browning rate and callus cell proliferation coefficient were calculated.

[0040] Table 4. Effects of different potato flour additions on callus growth of Saussurea edulis

[0041] The results show that callus culture in medium supplemented with 5 ppm of nisin z exhibited virtually no browning. Compared to medium 1, callus cells in medium supplemented with potato flour exhibited a more tender yellow color, a granular texture, a crisp and dense texture, and significant swelling. Adding 10-30 g of potato flour produced similar results. In summary, MS, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 10-30 g / L potato flour, 5 ppm nisin z, 30 g / L sucrose, and 3.5 g / L gellan gum are the optimal proliferation medium formulation for scale-up.

[0042] 6. Detection of active ingredients (1) Extraction of callus tissue To examine the effect of the aforementioned culture medium formulation on the active ingredients of Saussurea scoparia callus, sterile callus cultures with and without nisin Z (Table 4, number 4) were weighed for extraction. 50 g (wet weight) of each Saussurea scoparia callus were pulverized in a homogenizer three times for 2 minutes at a material-liquid ratio of 1:10-1:30. Extraction was then performed at 80°C for 3 hours, followed by centrifugation at 10,000 rpm for 60 minutes. The supernatant was filtered and freeze-dried to obtain a powdered Saussurea scoparia callus extract. The callus extract without nisin Z served as a blank control for active ingredient determination.

[0043] (2) Determination of total polyphenols Total polyphenol content was determined using the Folin-Ciocalteu method. Standard curve: Prepare a gallic acid solution at a concentration of 0.3300 mg / mL and dilute it sequentially to a concentration gradient of 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, and 0.3300 mg / mL. Prepare an aqueous solution of Saussurea scoparia callus extract powder. Add 500 μL of 10% Folin-Ciocalteu reagent, mix thoroughly, and let stand for 5 minutes (no more than 8 minutes). Then, add 400 μL of 7.5% Na₂CO₃ solution, mix thoroughly, and let it react at room temperature for 60 minutes. Finally, take 100 μL of the mixture and measure the absorbance at 765 nm on a microplate reader. The total polyphenol content of the Saussurea scoparia callus extract was calculated according to the standard curve, and the results are shown in Table 5: the total polyphenol content of the Saussurea scoparia callus extract cultured without nisin z was 1.73%, and the total polyphenol content of the Saussurea scoparia callus extract cultured with nisin z was 2.13%. The total polyphenol content of the Saussurea scoparia callus extract cultured with nisin z was 23.12% higher than that of the blank group.

[0044] (3) Determination of total flavonoids The aluminum nitrate colorimetric method was used to detect the total flavonoid content. Standard solution: Quercetin was used to prepare an ethanol solution of 0.2315 mg / mL, and diluted sequentially into a 2-10 times concentration gradient. The powder of the callus extract of Saussurea scoparia was prepared into an aqueous solution, 300 μL was taken, 50 μL of 5% NaNO2 solution was added first, mixed and allowed to stand at room temperature for 6 minutes; then 50 μL of 10% Al(NO3)3 solution was added, mixed and allowed to stand at room temperature for 6 minutes; finally, 500 μL of 4% NaOH and 140 μL of deionized water were added, mixed and allowed to stand at room temperature for 15 minutes. 100 μL of the mixed solution was taken and the absorbance at 500 nm was measured on an enzyme-linked microplate reader. The total flavonoid content of the Saussurea scoparia extract was calculated based on the standard curve. The results are shown in Table 5:

[0045] Table 5. Content of active ingredients in Saussurea involucrata callus

[0046] As can be seen from Table 5, the total flavonoids content of the extract of Saussurea scoparia callus cultured without adding nisin z is 2.44%, and the total flavonoids content of the extract of Saussurea scoparia callus cultured with adding nisin z is 3.39%. The total flavonoids content of the extract of Saussurea scoparia callus cultured with adding nisin z is 38.93% higher than that of the blank group.

[0047] In summary, the callus tissues of Saussurea edulis cultured without or with nisin z contained polyphenols and flavonoids. The use of nisin z to culture Saussurea edulis callus tissues could increase the content of total polyphenols and total flavonoids, with the increase rates of 23.12% and 38.93% respectively. 7. Antioxidant activity test ABTS free radical experiment ABTS generates a stable blue-green cationic free radical ABTS·+ under the action of potassium persulfate, which has a strong absorption peak at 734nm. When an antioxidant is present, electron transfer occurs with ABTS·+, reducing ABTS·+ to colorless ABTS, resulting in a decrease in the absorbance of the solution. The degree of decrease in absorbance is proportional to the antioxidant's ability to scavenge free radicals. ABTS solution is mixed with potassium persulfate and reacted for 12-16 hours at room temperature in the dark to prepare an ABTS·+ stock solution. Dilute with phosphate buffer or ethanol before use to an absorbance of 0.7±0.02 (734nm). Take a certain amount of sample solution and add the diluted ABTS Add the working solution, mix thoroughly, and react for 6 minutes. Measure the absorbance at 734 nm. Simultaneously, set up a control group (no sample). Calculate the ABTS free radical scavenging rate of the sample using the formula to assess its antioxidant activity. Vitamin C was used as a positive control.

[0048] The results are shown in Table 6

[0049] Table 6. Antioxidant activity of extracts from Saussurea scoparia callus

[0050] The positive control, vitamin C, had an ABTS scavenging rate of 99.82%. The ABTS scavenging rate of extracts from Saussurea scoparia calli cultured without nisin (the blank group) was 72.70%. The ABTS scavenging rate of extracts from Saussurea scoparia calli cultured with nisin was 91.24%. The total ABTS scavenging rate of extracts from Saussurea scoparia calli cultured with nisin was 18.54% higher than that of the blank group. In summary, extracts from Saussurea scoparia calli cultured with nisin exhibited higher antioxidant activity than extracts from uncultured Saussurea scoparia calli cultured with nisin.

Claims

1. A callus proliferation culture medium for Saussurea scoparia, characterized in that: It includes the following components: Basic medium for plant tissue culture, nisin 3-100 ppm; of which: Ppm refers to the concentration in the Saussurea involucrata callus proliferation culture medium.

2. The Saussurea involucrata callus proliferation culture medium according to claim 1, wherein The concentration of nisin Z is 4-80 ppm.

3. The Saussurea involucrata callus proliferation culture medium according to claim 1-2, wherein The basal culture medium is selected from MS culture medium, N6 culture medium, B5 culture medium, SH culture medium or White culture medium, preferably MS culture medium; And / or, the Saussurea involucrata callus proliferation culture medium further comprises plant hormones; the plant hormones are preferably selected from one or more of NAA, 6-BA, 2,4-D and KT; more preferably, 6-BA and NAA; The concentration of the plant hormone in the Saussurea involucrata callus proliferation culture medium is preferably 0.1-2.0 mg / L, more preferably 0.2-1.8 mg / L. When the plant hormones are 6-BA and NAA, the concentration of 6-BA in the Saussurea involucrata callus proliferation culture medium is 0.3-0.7 mg / L, and the concentration of NAA in the Saussurea involucrata callus proliferation culture medium is preferably 0.3-0.7 mg / L; And / or, the Saussurea involucrata callus proliferation culture medium further comprises a carbon source, such as one or more of sucrose, maltose and glucose, preferably sucrose; the concentration of the carbon source in the Saussurea involucrata callus proliferation culture medium is preferably 28-35 g / L, more preferably 30 g / L; And / or, the Saussurea scoparia callus proliferation culture medium further comprises a coagulant, such as agar and / or gellan gum, preferably gellan gum; the concentration of the coagulant in the Saussurea scoparia callus proliferation culture medium is preferably 3-4 g / L, more preferably 3.5 g / L; And / or, the Saussurea scoparia callus proliferation culture medium further comprises potato flour; the concentration of the potato flour in the Saussurea scoparia callus proliferation culture medium is 1-35 g / L, preferably 30 g / L.

4. A callus proliferation culture medium for Saussurea scoparia, characterized in that: The culture medium is MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + nisin z 5 ppm + sucrose 30 g / L + gellan gum 3.5 g / L + potato flour 30 g / L or MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + nisin z 50 ppm + sucrose 30 g / L + gellan gum 3.5 g / L + potato flour 30 g / L.

5. A method for preparing a culture medium for the proliferation of Saussurea involucrata callus, characterized in that: The method comprises the following steps: mixing the components in the Saussurea involucrata callus proliferation culture medium according to any one of claims 1 to 4.

6. A method for increasing the proliferation rate of Saussurea involucrata callus, characterized in that: The Saussurea scabra callus is cultured using the Saussurea scabra callus proliferation culture medium according to any one of claims 1 to 5.

7. The method according to claim 6, wherein The callus of Saussurea scabra is induced by the following method: the seeds of wild Saussurea scabra are sterilized and placed in MS basal medium in the dark for cultivation, and after the seeds germinate and grow to about 1 cm, they are transferred to MS culture bottles for cultivation to obtain sterile seedlings; The roots, stems, and leaves of the sterile seedlings were cut into segments of 0.5 cm to 1.0 cm in length and inoculated into an induction medium of MS + 0.5 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 3.5 g / L gellan gum. After dark culture, dense tissue appeared at the incision. After continuing to culture for 30 days, the cotton callus tissue was obtained.

8. A callus cell extract of Saussurea scoparia, characterized in that: The method is obtained by extracting cultured Saussurea scoparia callus cells, wherein the cultured Saussurea scoparia callus cells are obtained by culturing Saussurea scoparia callus tissue using the Saussurea scoparia callus proliferation culture medium according to any one of claims 1 to 4.

9. The Saussurea involucrata callus cell extract according to claim 8, wherein The culture conditions are: temperature 20±1°C, humidity 50%~70%, dark culture; And / or, the content of polyphenols in the Saussurea scoparia callus cell extract is 2-2.2%; the content of polyphenols refers to the mass percentage of polyphenols in the Saussurea scoparia callus cell extract; And / or, the content of flavonoids in the Saussurea scoparia callus cell extract is 3-3.5%.

10. Use of an extract of Saussurea involucrata callus cells in the preparation of a skin external preparation, characterized in that: The Saussurea scabra callus cell extract according to claim 8 is used as the skin external preparation, antioxidant active ingredient, anti-aging active ingredient or whitening active ingredient in health food.

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