A method for culturing rhodiola rosea stem cells

Abstract

The present invention provides a method for culturing Rhodiola rosea stem cells, the method comprising subjecting the stem cells derived from the roots or root tips of sterile seedlings germinated from seeds to multiple generations of screening and amplification to obtain a Rhodiola rosea stem cell mass, culturing the stem cell mass in suspension and then inducing the mass in a liquid culture medium preferably under LED irradiation conditions to obtain Rhodiola rosea stem cells containing more whitening and antioxidant active ingredients. The present invention also relates to a method for preparing a Rhodiola rosea stem cell extract. The present invention also relates to a Rhodiola rosea stem cell extract. The present invention further relates to the use of the Rhodiola rosea stem cell extract in the preparation of medicines or cosmetics and medicines or cosmetics containing the Rhodiola rosea stem cell extract.

Description

Technical Field

[0001] The present invention relates to a method for culturing Rhodiola rosea stem cells. The present invention also relates to a method for preparing a Rhodiola rosea stem cell extract. The present invention also relates to a Rhodiola rosea stem cell extract. The present invention further relates to the use of the Rhodiola rosea stem cell extract in the preparation of a medicine or cosmetic and a medicine or cosmetic containing the Rhodiola rosea stem cell extract. Background Art

[0002] Rhodiola crenulate is a plant with extremely high medicinal value, and its roots and rhizomes are widely used in traditional Chinese medicine. According to the "Shennong Bencao Jing", Rhodiola crenulate is listed as a top-grade medicinal material, with the effects of invigorating qi and activating blood circulation, unblocking meridians and relieving asthma. It is often used to treat qi deficiency and blood stasis, chest pain, heart pain, stroke hemiplegia, fatigue and asthma. Modern pharmacological research has further revealed the various pharmacological activities of Rhodiola crenulate, including anti-hypoxia, anti-fatigue, anti-aging, anti-virus, anti-tumor, etc.

[0003] At present, the medicinal resources of Rhodiola rosea are completely dependent on wild resources, which are mainly distributed in the high-altitude and cold areas in western China, such as Tibet, Qinghai, Sichuan and Yunnan. Due to its harsh growth environment and low reproduction rate, wild resources are at risk of depletion and have been listed as a national second-level protected wild plant. In order to alleviate the problem of resource shortage, some regions have begun to explore rapid propagation technologies for Rhodiola rosea, such as artificial propagation, tissue culture, and plant stem cell technology. However, these technologies still have some problems in production practice, such as slow growth rate of artificial propagation, low survival rate of transplantation of tissue culture regenerated seedlings, and low content of active substances in Rhodiola rosea stem cells, which has made it impossible to use plant rapid propagation technology to achieve industrialized propagation of Rhodiola rosea.

[0004] The existing Rhodiola rosea stem cell culture technology still has the following problems or shortcomings: (1) The existing Rhodiola rosea stem cell culture technology generally extracts stem cells from the cambium of the roots. A series of enzymes are added during the extraction process. The enzymes may decompose the pharmacologically active substances during the process of enzymatic hydrolysis of the cell wall, thereby reducing the pharmacological effects of Rhodiola rosea stem cells; (2) Most of the current Rhodiola rosea stem cell culture technologies are limited to the extraction and culture of stem cells, and lack a culture method for the targeted production of pharmacologically active substances, resulting in low activity of Rhodiola rosea stem cell extracts.

[0005] Based on the above technical problems existing in the prior art, the present invention has established a method for screening, culturing and accumulating active ingredients of Rhodiola rosea stem cells, which has a short time period and does not require a large amount of land resources. The obtained stem cell extract is rich in pharmacologically active substances, which helps to make up for the current problems of complete reliance on wild resources and insufficient wild resources, and is of great significance to solving the problem of medicinal resources of Rhodiola rosea. Summary of the invention

[0006] The present invention relates to a method for culturing Rhodiola rosea stem cells, the method comprising: screening and amplifying stem cells from the roots or root tips of sterile seedlings germinated from seeds for multiple generations to obtain Rhodiola rosea stem cell clusters, culturing the stem cell clusters in suspension and then inducing them in a liquid culture medium under LED irradiation conditions to obtain Rhodiola rosea stem cells containing more whitening and antioxidant active ingredients. The method for culturing Rhodiola rosea stem cells of the present invention has the following advantages:

[0007] (1) Compared with wild growth and reproduction of Rhodiola rosea, the time cycle is short and does not require a large amount of land resources, which helps to make up for the current problems of complete reliance on wild resources and insufficient wild resources, and is of great significance to solving the problem of medicinal resources of Rhodiola rosea;

[0008] (2) The present invention screens stem cells from the roots of sterile seedlings germinated from seeds, and obtains Rhodiola rosea stem cells with self-renewal and replication ability, differentiation potential, stable growth, rapid proliferation, and long-term stable culture after multiple generations of screening;

[0009] (3) By using a combination of multiple culture conditions and activity-guided screening, the optimal culture conditions for the extract to have significant whitening and antioxidant effects were obtained;

[0010] (4) The Rhodiola rosea stem cell line is stable and does not require the addition or combination of other enzymes or cells to exert whitening and antioxidant effects;

[0011] (5) This cell culture method is easy to operate, saves time and effort, and does not require frequent replacement of culture medium;

[0012] (6) In the prior art, the Rhodiola rosea stem cell culture technology usually extracts stem cells from the cambium of the root, and the process requires the use of some enzymes to enzymatically hydrolyze the cell wall of the Rhodiola rosea root. However, the process of the present invention extracting stem cells from the root or root tip of sterile seedlings germinated from seeds does not require the enzymatic hydrolysis process, thereby avoiding the situation where the enzyme may decompose the pharmacologically active substances in the Rhodiola rosea stem cells, thereby reducing the pharmacological effects of the Rhodiola rosea stem cells.

[0013] The first aspect of the present invention relates to a method for culturing Rhodiola rosea stem cells, the method comprising the following steps:

[0014] (1) inoculating sterile Rhodiola rosea seeds and culturing them until they germinate and grow into seedlings;

[0015] (2) isolating the roots or root tips of the seedlings in step (1), culturing Rhodiola rosea stem cells on a solid culture medium, and further amplifying the cultured Rhodiola rosea stem cells to obtain stem cell aggregates;

[0016] (3) transferring the stem cell mass formed in step (2) to a liquid culture medium for suspension culture for a period of time until the stem cells are fully adapted to the liquid culture conditions and can stably reproduce in the liquid culture medium;

[0017] (4) Inoculating the stem cells cultured in suspension in step (3) into a liquid culture medium, and culturing under conditions selected from a dark room, LED illumination, fluorescent illumination, and a combination thereof, preferably culturing under LED illumination.

[0018] The second aspect of the present invention relates to a method for culturing Rhodiola rosea stem cells, the method comprising the following steps:

[0019] (1) inoculating sterile Rhodiola rosea seeds and culturing them until they germinate and grow into seedlings;

[0020] (2) isolating the roots or root tips of the seedlings in step (1), culturing Rhodiola rosea stem cells on a solid culture medium, and further amplifying the cultured Rhodiola rosea stem cells to obtain stem cell aggregates; and

[0021] (5) Transferring the stem cells obtained in step (2) to a solid culture medium, and culturing them under conditions selected from a dark room, LED illumination, fluorescent illumination, and a combination thereof, preferably culturing them under LED illumination.

[0022] The third aspect of the present invention relates to a method for preparing a Rhodiola rosea stem cell extract, which comprises extracting Rhodiola rosea stem cells cultured according to the culture method described in the first aspect or the second aspect of the present invention.

[0023] The fourth aspect of the present invention relates to a Rhodiola rosea stem cell extract, which is prepared by the method described in the third aspect of the present invention.

[0024] The fifth aspect of the present invention relates to use of the Rhodiola rosea stem cell extract according to the fourth aspect of the present invention in the preparation of medicines or cosmetics.

[0025] The sixth aspect of the present invention relates to a medicine or cosmetic comprising the Rhodiola rosea stem cell extract according to the fourth aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Graphs showing seed germination and seedling growth during an experiment according to Example 1 of the present application.

[0027] Figure 2 A diagram showing the induction of primary cells in a solid culture medium from the shoot tip, root tip, leaf, stem segment and root segment of a seedling according to Example 2 of the present application.

[0028] Figure 3The figure shows the white callus cell masses obtained after the stem tips, root tips, leaves, stem segments and root segments of the seedlings according to Example 2 of the present application were selected and cultured in a solid culture medium for 2-5 months.

[0029] Figure 4 A diagram showing cell aggregates obtained after expansion culture in a solid medium according to Example 2 of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition

[0032] As used herein, the terms "s", "min" and "h" stand for "seconds", "minutes" and "hours", respectively.

[0033] As used herein, the term "comprising", which is synonymous with "including", "containing" or "characterized by", is inclusive or open-ended and does not exclude additional, undescribed elements or method steps. However, in the text, each time "comprising" is mentioned, it is intended to cover the alternative embodiments of "consisting essentially of" and "consisting of", wherein "consisting of" excludes any elements or steps not specified, and "consisting essentially of" allows the inclusion of other elements or steps not described that do not materially affect the essential or basic characteristics and novel characteristics of the composition or method in question.

[0034] The term "about" as used herein refers to ±10%, more preferably ±5%, and most preferably ±2% of the value modified by the term, so that a person skilled in the art can clearly determine the scope of the term "about" based on the modified value.

[0035] The term "inducer" as used herein refers to a substance that can promote the synthesis and accumulation of secondary metabolites in cells during the tissue culture process of Rhodiola rosea stem cells.

[0036] Cultivation method of Rhodiola rosea stem cells

[0037] The first aspect of the present invention relates to a method for culturing Rhodiola rosea stem cells, the method comprising the following steps:

[0038] (1) inoculating sterile Rhodiola rosea seeds and culturing them until they germinate and grow into seedlings;

[0039] (2) isolating the roots or root tips of the seedlings in step (1), culturing Rhodiola rosea stem cells on a solid culture medium, and further amplifying the cultured Rhodiola rosea stem cells to obtain stem cell aggregates;

[0040] (3) transferring the stem cell mass formed in step (2) to a liquid culture medium for suspension culture for a period of time until the stem cells are fully adapted to the liquid culture conditions and can stably reproduce in the liquid culture medium;

[0041] (4) Inoculating the stem cells cultured in suspension in step (3) into a liquid culture medium, and culturing under conditions selected from a dark room, LED illumination, fluorescent illumination, and a combination thereof, preferably culturing under LED illumination.

[0042] The second aspect of the present invention relates to a method for culturing Rhodiola rosea stem cells, the method comprising the following steps:

[0043] (1) inoculating sterile Rhodiola rosea seeds and culturing them until they germinate and grow into seedlings;

[0044] (2) isolating the roots or root tips of the seedlings in step (1), culturing Rhodiola rosea stem cells on a solid culture medium, and further amplifying the cultured Rhodiola rosea stem cells to obtain stem cell aggregates; and

[0045] (5) Transferring the stem cells obtained in step (2) to a solid culture medium, and culturing them under conditions selected from a dark room, LED illumination, fluorescent illumination, and a combination thereof, preferably culturing them under LED illumination.

[0046] About step (1)

[0047] In some embodiments, the Rhodiola rosea is selected from the group consisting of Rhodiola angusta, Rhodiola rosea, Rhodiola sacra, Rhodiola calliantha (H. Ohba) H. Ohba, Rhodiola cretinii (Raym.-Hamet) H. Ohba, Rhodiola heterodonta (Hook.f. & Thomson) Boriss., Rhodiola kirilowii (Regel) Maxim., Rhodiola linearifolia A. Bor., Rhodiola akirilowii (Regel) Maxim., Rhodiola rosea L., Rhodiola serrata H. Ohba, Rhodiola dumulosa (Franch.) SHFu), Rhodiola angusta, Rhodiola fastigiate, Rhodiola quadrifida, Rhodiola sachalinensis, Rhodiola tangutica, Rhodiola wallichiana, Rhodiola yunnanensis and Rhodiola himalensis, preferably Rhodiola grandiflora

[0048] In some embodiments, in step (1), the seeds are sterilized by a treatment selected from ethanol treatment, sodium hypochlorite treatment, sterile water washing and a combination thereof. In some embodiments, in step (1), the seeds are sterilized by ethanol treatment, sodium hypochlorite treatment and sterile water washing. In some embodiments, in step (1), the seeds are sterilized by sequentially performing ethanol treatment, sodium hypochlorite treatment and sterile water washing. In some embodiments, each of the ethanol treatment, sodium hypochlorite treatment and sterile water washing can be performed once or multiple times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc. In some embodiments, after the ethanol treatment or sodium hypochlorite treatment, the ethanol used for the treatment or the sodium hypochlorite used for the treatment is removed respectively.

[0049] In some embodiments, the ethanol treatment is performed using 60%-90% ethanol, preferably 70%-80% ethanol, and most preferably 75% ethanol. In some embodiments, the ethanol treatment is performed for 20-60 seconds. In some embodiments, after each ethanol treatment, the ethanol used in the treatment is removed by suction.

[0050] In some embodiments, the sodium hypochlorite treatment is a treatment using 5%-15%, preferably 10% sodium hypochlorite. In some embodiments, the sodium hypochlorite treatment time is 3-10 minutes. In some embodiments, after each sodium hypochlorite treatment, the sodium hypochlorite used in the treatment is sucked off. In some embodiments, the seed sample to which the sodium hypochlorite solution is added is repeatedly shaken during the sodium hypochlorite treatment.

[0051] In some embodiments, the sterile water flushing process is performed 3-6 times. In some embodiments, the sterile water flushing process is performed 3 times, 4 times, 5 times or 6 times.

[0052] In some embodiments, in step (1), the sterile seeds are first inoculated in MS medium and cultured under 8-16 hours / day of trichromatic fluorescent light until the seeds germinate; then the germinated seeds are transferred to modified White solid medium and cultured under 8-16 hours / day of full-spectrum LED light until seedlings grow.

[0053] In some embodiments, the MS medium is a commercially available MS medium.

[0054] MS medium, the full name of which is Murashige and Skoog medium, was designed by Murashige and Skoog in 1962. In a specific embodiment of the present invention, its formula composition is: 1650mg / L NH4NO3, 332.2mg / L CaCl2, 180.7mg / L MgSO4, 1900mg / L KNO3, 170mg / L KH2PO4, 6.2mg / L H3BO3, 0.025mg / LCoCl2·6H2O, 0.025mg / L CuSO4·5H2O, 36.7mg / L FeNaEDTA, 16.9mg / L MnSO4·H2O, 0.25mg / L Na2MoO4·2H2O, 0.83mg / L KI, 8.6 mg / L ZnSO4·7H2O, 2 mg / L glycine, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 0.5 mg / L pyridoxine hydrochloride, 0.1 mg / L thiamine hydrochloride and appropriate amounts of sucrose and agar.

[0055] In some embodiments, the MS medium used in step (1) is an MS medium comprising 3-10 g / L agar and 20-40 g / L sucrose. In some embodiments, the solid medium used in step (2) is an MS medium comprising 5.5 g / L agar and 30 g / L sucrose.

[0056] In some embodiments, the intensity of the three-primary-color fluorescent light used in step (1) is 3000-4000 lux. In some embodiments, the three-primary-color fluorescent light illumination time used in step (1) is 8-16 hours / day. In some embodiments, the three-primary-color fluorescent light illumination time used in step (1) is 9-11 hours / day. In some embodiments, the three-primary-color fluorescent light illumination time used in step (1) is 10 hours / day.

[0057] In some embodiments, the temperature at which the sterile-treated seeds are cultured in the MS medium is 20° C. to 30° C. In some embodiments, the temperature at which the sterile-treated seeds are cultured in the MS medium is 23° C. to 25° C. In some embodiments, the temperature at which the sterile-treated seeds are cultured in the MS medium is 20° C. to 21° C., 21° C. to 22° C., 22° C. to 23° C., 23° C. to 24° C., 24° C. to 25° C., 25° C. to 26° C., 26° C. to 27° C., 27° C. to 28° C., 28° C. to 29° C., or 29° C. to 30° C.

[0058] In some embodiments, the time for culturing the aseptically treated seeds in the MS medium is 20 to 40 days. In some embodiments, the time for culturing the aseptically treated seeds in the MS medium is 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days or 40 days. In some embodiments, the time for culturing the aseptically treated seeds in the MS medium is 30 days.

[0059] In some embodiments, the modified White's solid medium is a commercially available modified White's solid medium.

[0060] The formula of the improved White solid medium is: 65mg / L KCl, 80mg / L KNO3, 351.6mg / L MgSO4, 16.8mg / L NaH2PO4, 0.001mg / L CuSO4·5H2O, 3.47mg / L FeSO4·7H2O, 1.5H3BO3, 0.75mg / LKI, 5.31mg / LMnSO4·H2O, 0.0001mg / L MoO3, 200mg / L Na2SO4, 2.67mg / LZnSO4·7H2O, 2mg / L glycine, 100mg / L inositol, 0.1mg / L thiamine hydrochloride, 0.5mg / L niacin, 0.5mg / L pyridoxine hydrochloride, 300mg / L Ca(NO3)2·4H2O and appropriate amounts of sucrose and agar.

[0061] In some embodiments, the modified White solid medium used in step (1) is an MS medium comprising 3-10 g / L agar and 20-40 g / L sucrose. In some embodiments, the solid medium used in step (2) is an MS medium comprising 5.5 g / L agar and 30 g / L sucrose.

[0062] In some embodiments, the intensity of the full-spectrum LED illumination used in step (1) is 6000-7000 lux. In some embodiments, the full-spectrum LED illumination time used in step (1) is 8-16 hours / day. In some embodiments, the full-spectrum LED illumination time used in step (1) is 11-13 hours / day. In some embodiments, the full-spectrum LED illumination time used in step (1) is 12 hours / day.

[0063] In some embodiments, the temperature at which the aseptically treated seeds are cultured in the modified white solid medium is 20° C. to 30° C. In some embodiments, the temperature at which the aseptically treated seeds are cultured in the modified white solid medium is 23° C. to 25° C. In some embodiments, the temperature at which the aseptically treated seeds are cultured in the modified white solid medium is 20° C. to 21° C., 21° C. to 22° C., 22° C. to 23° C., 23° C. to 24° C., 24° C. to 25° C., 25° C. to 26° C., 26° C. to 27° C., 27° C. to 28° C., 28° C. to 29° C., or 29° C. to 30° C.

[0064] In some embodiments, the time of aseptically treated seeds in the modified white solid medium is 50 to 100 days. In some embodiments, the time of aseptically treated seeds in the modified white solid medium is 50 to 55 days, 55 to 60 days, 60 to 65 days, 65 to 70 days, 70 to 75 days, 75 to 80 days, 80 to 85 days, 85 to 90 days, 90 to 95 days or 95 to 100 days. In some embodiments, the time of aseptically treated seeds in the modified white solid medium is 60 to 90 days.

[0065] In some embodiments, step (1) includes the following specific processes: treating Rhodiola rosea seeds with ethanol for 20-60 seconds and absorbing the ethanol; adding sodium hypochlorite and shaking repeatedly for 3-10 minutes; after absorbing the sodium hypochlorite, washing with sterile water for 3-6 times to obtain sterile seeds; inoculating the sterile seeds on MS solid culture medium; irradiating with trichromatic fluorescent light (3000 to 4000 lux) for 8-16 hours / day, at a temperature of 20°C to 30°C, and culturing for 20 to 40 days; selecting seedlings with 2-3 leaves, transferring them to modified White solid culture medium, irradiating with full-spectrum LED light (6000 to 7000 lux) for 8-16 hours / day, at a temperature of 20°C to 30°C, and culturing for 50 to 100 days.

[0066] About step (2)

[0067] In some embodiments, in step (2), the roots or root tips of the seedlings separated in step (1) are cultured in the solid culture medium in step (2) until white dot-shaped Rhodiola rosea stem cells are produced on the tissue, and then the white dot-shaped Rhodiola rosea stem cells are separated and inoculated separately into another solid culture medium of the same composition for screening and culture, and the screened Rhodiola rosea stem cells that can stably reproduce are amplified and cultured, and after multiple generations of amplification and culture, white loose stem cell clusters are obtained.

[0068] In some embodiments, the solid culture medium used in step (2) is an MS culture medium containing 6-benzylaminopurine and naphthaleneacetic acid. In some embodiments, the solid culture medium used in step (2) is an MS culture medium containing 0.5-2.0 mg / L of 6-benzylaminopurine and 0.1-1.0 mg / L of naphthaleneacetic acid. In some embodiments, the solid culture medium used in step (2) is an MS culture medium containing 1 mg / L of 6-benzylaminopurine and 0.2 mg / L of naphthaleneacetic acid.

[0069] In some embodiments, the solid culture medium used in step (2) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid, agar and sucrose. In some embodiments, the solid culture medium used in step (2) is an MS culture medium comprising 0.5-2.0mg / L 6-benzylaminopurine, 0.1-1.0mg / L naphthaleneacetic acid, 3-10g / L agar and 20-40g / L sucrose. In some embodiments, the solid culture medium used in step (2) is an MS culture medium comprising 1mg / L 6-benzylaminopurine, 0.2mg / L naphthaleneacetic acid, 5.5g / L agar and 30g / L sucrose.

[0070] In some embodiments, the solid culture medium used in step (2) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid, agar and sucrose, wherein the concentration of 6-benzylaminopurine in the culture medium is 0.5-2mg / L, preferably 0.8-1.2mg / L, and most preferably 1mg / L. In some embodiments, the solid culture medium used in step (2) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid, agar and sucrose, wherein the concentration of naphthaleneacetic acid in the culture medium is 0.1-0.5mg / L, preferably 0.1-0.3mg / L, and most preferably 0.2mg / L. In some embodiments, the solid culture medium used in step (2) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid, agar and sucrose, wherein the concentration of agar in the culture medium is 3-10g / L, preferably 5-6g / L, and most preferably 5.5g / L. In some embodiments, the solid culture medium used in step (2) is an MS medium comprising 6-benzylaminopurine, naphthylacetic acid, agar and sucrose, wherein the concentration of sucrose in the culture medium is 20-40 g / L, preferably 25-35 g / L, and most preferably 30 g / L.

[0071] In some embodiments, the solid culture medium used in step (2) is an MS culture medium in which, except for the contents of 6-benzylaminopurine, naphthylacetic acid, agar and sucrose as defined above, the contents of other components are the same as the contents of corresponding components in commercially available MS culture medium. In some embodiments, the solid culture medium used in step (2) is as follows: 1650 mg / L NH4NO3, 332.2 mg / L CaCl2, 180.7 mg / L MgSO4, 1900 mg / L KNO3, 170 mg / L KH2PO4, 6.2 mg / L H3BO3, 0.025 mg / L CoCl2·6H2O, 0.025 mg / L CuSO4·5H2O, 36.7 mg / L FeNaEDTA, 16.9 mg / L MnSO4·H2O, 0.25 mg / L Na2MoO4·2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 2 mg / L glycine, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride.

[0072] In some embodiments, in the solid culture medium used in step (2), the mass ratio of 6-benzylaminopurine to naphthaleneacetic acid is 1: 1 to 10: 1. In some embodiments, in the solid culture medium used in step (2), the mass ratio of 6-benzylaminopurine to naphthaleneacetic acid is 1: 1 to 2: 1, 2: 1 to 3: 1, 3: 1 to 4: 1, 4: 1 to 5: 1, 5: 1 to 6: 1, 6: 1 to 7: 1, 7: 1 to 8: 1, 8: 1 to 9: 1 or 9: 1 to 10: 1. In some embodiments, in the solid culture medium used in step (2), the mass ratio of 6-benzylaminopurine to naphthaleneacetic acid is 5: 1.

[0073] In some embodiments, the culturing in step (2) is performed under dark room conditions.

[0074] In some embodiments, the temperature of the culture in the solid medium in step (2) is 20° C. to 30° C. In some embodiments, the temperature of the culture in the solid medium in step (2) is 23° C. to 25° C. In some embodiments, the temperature of the culture in the solid medium in step (2) is 20° C. to 21° C., 21° C. to 22° C., 22° C. to 23° C., 23° C. to 24° C., 24° C. to 25° C., 25° C. to 26° C., 26° C. to 27° C., 27° C. to 28° C., 28° C. to 29° C., or 29° C. to 30° C.

[0075] In some embodiments, in step (2), the roots or root tips of the seedlings separated in step (1) are cultured in the solid culture medium in step (2) for 20-30 days until white dot-like Rhodiola rosea stem cells are produced on the tissue, and then the white dot-like Rhodiola rosea stem cells are separated and inoculated separately into another solid culture medium of the same composition for expansion culture for 2-5 months, and after multiple generations of expansion culture, white loose stem cell clusters are obtained.

[0076] In some embodiments, step (2) includes the following specific processes: separating the roots or root tips of the seedlings in step (1), inoculating them onto a solid culture medium, and culturing them in a dark room at a temperature of 20°C to 30°C for 20-30 days until white raised dot-shaped Rhodiola rosea stem cells are produced on the tissue; then separating the white raised dot-shaped Rhodiola rosea stem cells and inoculating them separately into another solid culture medium of the same composition for expansion culture for 2-5 months, and obtaining white loose stem cell clusters after multiple generations of expansion culture; optionally, culturing the cells separated from the white loose stem cell clusters in a dark room at a temperature of 20°C to 30°C for 60-90 days.

[0077] About step (3)

[0078] In some embodiments, in step (3), the stem cell cluster formed in step (2) is first transferred to a liquid culture medium of volume A and suspended and cultured at a rotation speed M for a period of time until no browning occurs, and then transferred to a liquid culture medium of volume B and suspended and cultured at a rotation speed N for a period of time, wherein the volume A is smaller than the volume B, and the rotation speed M is smaller than the rotation speed N.

[0079] In some embodiments, in step (3), volume A is 50-100 mL. In some embodiments, volume A is 50-55 mL, 55-60 mL, 60-65 mL, 65-70 mL, 70-75 mL, 75-80 mL, 80-85 mL, 85-90 mL, 90-95 mL or 95-100 mL. In some embodiments, in step (3), volume A is 70 mL. In some embodiments, in step (3), volume A is an amount that allows the cell portion to be exposed to the air.

[0080] In some embodiments, in step (3), the rotation speed M is 50-100 rpm. In some embodiments, in step (3), the rotation speed M is 50-55 rpm, 55-60 rpm, 60-65 rpm, 65-70 rpm, 70-75 rpm, 75-80 rpm, 80-85 rpm, 85-90 rpm, 90-95 rpm or 95-100 rpm. In some embodiments, in step (3), the rotation speed M is 70 rpm.

[0081] In some embodiments, in step (3), volume B is 150-250 mL. In some embodiments, volume B is 150-160 mL, 160-170 mL, 170-180 mL, 180-190 mL, 190-200 mL, 200-210 mL, 210-220 mL, 220-230 mL, 230-240 mL or 240-250 mL. In some embodiments, in step (3), volume B is 200 mL.

[0082] In some embodiments, in step (3), the rotation speed N is 100-150 rpm. In some embodiments, in step (3), the rotation speed N is 100-105 rpm, 105-110 rpm, 110-115 rpm, 115-120 rpm, 120-125 rpm, 125-130 rpm, 130-135 rpm, 135-140 rpm, 140-145 rpm or 145-150 rpm. In some embodiments, in step (3), the rotation speed N is 120 rpm.

[0083] In some embodiments, the culturing in step (3) is performed under dark room conditions.

[0084] In some embodiments, the temperature of the culture in the liquid culture medium in step (3) is 20° C. to 30° C. In some embodiments, the temperature of the culture in the liquid culture medium in step (3) is 23° C. to 25° C. In some embodiments, the temperature of the culture in the liquid culture medium in step (3) is 20° C. to 21° C., 21° C. to 22° C., 22° C. to 23° C., 23° C. to 24° C., 24° C. to 25° C., 25° C. to 26° C., 26° C. to 27° C., 27° C. to 28° C., 28° C. to 29° C., or 29° C. to 30° C.

[0085] In some embodiments, the liquid culture medium used in step (3) is an MS culture medium containing 6-benzylaminopurine and naphthaleneacetic acid and not containing agar. In some embodiments, the liquid culture medium used in step (3) is an MS culture medium containing 0.5-2.0 mg / L of 6-benzylaminopurine and 0.1-1.0 mg / L of naphthaleneacetic acid and not containing agar. In some embodiments, the liquid culture medium used in step (3) is an MS culture medium containing 1 mg / L of 6-benzylaminopurine and 0.2 mg / L of naphthaleneacetic acid and not containing agar.

[0086] In some embodiments, the liquid culture medium used in step (3) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid and sucrose and not comprising agar. In some embodiments, the liquid culture medium used in step (3) is an MS culture medium comprising 0.5-2.0 mg / L of 6-benzylaminopurine, 0.1-1.0 mg / L of naphthaleneacetic acid and 20-40 g / L of sucrose and not comprising agar. In some embodiments, the liquid culture medium used in step (3) is an MS culture medium comprising 1 mg / L of 6-benzylaminopurine, 0.2 mg / L of naphthaleneacetic acid and 30 g / L of sucrose and not comprising agar.

[0087] In some embodiments, the liquid culture medium used in step (3) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid and sucrose and not comprising agar, wherein the concentration of 6-benzylaminopurine in the culture medium is 0.5-2 mg / L, preferably 0.8-1.2 mg / L, and most preferably 1 mg / L. In some embodiments, the liquid culture medium used in step (3) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid and sucrose and not comprising agar, wherein the concentration of naphthaleneacetic acid in the culture medium is 0.1-0.5 mg / L, preferably 0.1-0.3 mg / L, and most preferably 0.2 mg / L. In some embodiments, the liquid culture medium used in step (3) is an MS culture medium comprising 6-benzylaminopurine, naphthaleneacetic acid and sucrose and not comprising agar, wherein the concentration of sucrose in the culture medium is 20-40 g / L, preferably 25-35 g / L, and most preferably 30 g / L.

[0088] In some embodiments, the liquid culture medium used in step (3) is an MS culture medium in which the contents of the components, except for 6-benzylaminopurine, naphthylacetic acid and sucrose, are as defined above and agar is not included, and the contents of the other components are the same as those of the corresponding components in the commercially available MS culture medium. In some embodiments, the liquid culture medium used in step (3) is as defined above except that the contents of 6-benzylaminopurine, naphthylacetic acid and sucrose are not included in the agar, and the contents of the other components are as follows: 1650 mg / L NH4NO3, 332.2 mg / L CaCl2, 180.7 mg / L MgSO4, 1900 mg / L KNO3, 170 mg / L KH2PO4, 6.2 mg / L H3BO3, 0.025 mg / L CoCl2·6H2O, 0.025 mg / L CuSO4·5H2O, 36.7 mg / L FeNaEDTA, 16.9 mg / L MnSO4·H2O, 0.25 mg / L Na2MoO4·2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 2 mg / L glycine, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride.

[0089] In some embodiments, in the liquid culture medium used in step (3), the mass ratio of 6-benzylaminopurine to naphthaleneacetic acid is 1: 1 to 10: 1. In some embodiments, in the liquid culture medium used in step (3), the mass ratio of 6-benzylaminopurine to naphthaleneacetic acid is 1: 1 to 2: 1, 2: 1 to 3: 1, 3: 1 to 4: 1, 4: 1 to 5: 1, 5: 1 to 6: 1, 6: 1 to 7: 1, 7: 1 to 8: 1, 8: 1 to 9: 1 or 9: 1 to 10: 1. In some embodiments, in the liquid culture medium used in step (3), the mass ratio of 6-benzylaminopurine to naphthaleneacetic acid is 5: 1.

[0090] In some embodiments, in step (3), the stem cell mass formed in step (2) is first transferred to a liquid culture medium of volume A and cultured in suspension at a rotation speed M for 2-5 weeks until no browning occurs, and then transferred to a liquid culture medium of volume B and cultured in suspension at a rotation speed N for 20-30 days, wherein the volume A is smaller than the volume B, and the rotation speed M is smaller than the rotation speed N.

[0091] In some embodiments, step (3) includes the following specific process: transferring the stem cells obtained in step (2) into 50-100 mL of liquid culture medium, exposing the cells partially to air, and shaking and culturing at 50-100 rpm in a dark room for 2-5 weeks; then transferring the stem cells into 150-250 mL of liquid culture medium, and shaking and culturing at 100-150 rpm in a dark room for 20-30 days.

[0092] About step (4)

[0093] In some embodiments, in step (4), the stem cells suspended and cultured in step (3) are inoculated into a liquid culture medium and cultured under conditions selected from a darkroom, LED lighting, fluorescent lighting, and a combination thereof, preferably under LED lighting. In some embodiments, the illumination intensity of the LED lighting in step (4) is 2000 to 6000 lux. In some embodiments, the illumination intensity of the LED lighting in step (4) is 2000 to 2200 lux, 2200 to 2400 lux, 2400 to 2600 lux, 2600 to 2800 lux, 2800 to 3000 lux, 3000 to 3200 lux, 3200 to 3400 lux, 3400 to 3600 lux, 3600 to 3800 lux, In some embodiments, the illumination intensity of the LED illumination in step (4) is 3400 to 5000 lux.

[0094] In some embodiments, in step (4), the LED illumination or the fluorescent illumination is optionally accompanied by irradiation of a 5000 to 8000K LED lamp as an external light source. In some embodiments, in step (4), the LED illumination or the fluorescent illumination is optionally accompanied by irradiation of a 5000 to 5500K, 5500 to 6000K, 6000 to 6500K, 6500 to 7000K, 7000 to 7500K or 7500 to 8000K LED lamp as an external light source.

[0095] In some embodiments, in step (4), the LED illumination or the fluorescent illumination is not accompanied by illumination with a 5000 to 8000K LED lamp as external light.

[0096] In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 8-16 hours / day. In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 8-9 hours / day, 9-10 hours / day, 10-11 hours / day, 11-12 hours / day, 12-13 hours / day, 13-14 hours / day, 14-15 hours / day or 15-16 hours / day. In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 11-13 hours / day. In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 12 hours / day.

[0097] In some embodiments, in step (4), darkroom culture, LED light culture and / or fluorescence culture are performed alternately. In some embodiments, in step (4), stem cell culture is performed simultaneously under LED light and fluorescence light.

[0098] In some embodiments, the culture time in step (4) is 21-42 days. In some embodiments, the culture time in step (4) is 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days or 42 days. In some embodiments, the culture time in step (4) is 23-31 days. In some embodiments, the culture time in step (4) is 28 days.

[0099] In some embodiments, optionally, an inducer is added during the culturing process of step (4). In some embodiments, the inducer is selected from methyl jasmonate, yeast elicitor, salicylic acid or a combination thereof.

[0100] In some embodiments, when methyl jasmonate is added as an inducer during the culture process of step (4), the final concentration of the added methyl jasmonate in the culture medium is 100 to 300 μM / L. In some embodiments, when methyl jasmonate is added as an inducer during the culture process of step (4), the final concentration of the added methyl jasmonate in the culture medium is 100 to 150 μM / L, 150 to 200 μM / L, 200 to 250 μM / L, or 250 to 300 μM / L. In some embodiments, when methyl jasmonate is added as an inducer during the culture process of step (4), the final concentration of the added methyl jasmonate in the culture medium is 200 μM / L.

[0101] In some embodiments, when a yeast elicitor is added as an inducer during the culture process of step (4), the final concentration of the added yeast elicitor in the culture medium is 0.5 to 2.0 g / L. In some embodiments, when a yeast elicitor is added as an inducer during the culture process of step (4), the final concentration of the added yeast elicitor in the culture medium is 0.5 to 0.75 g / L, 0.75 to 1.0 g / L, 1.0 to 1.25 g / L, 1.25 to 1.5 g / L, 1.5 to 1.75 g / L or 1.75 to 2.0 g / L. In some embodiments, when a yeast elicitor is added as an inducer during the culture process of step (4), the final concentration of the added methyl jasmonate in the culture medium is 1.25 g / L.

[0102] In some embodiments, when salicylic acid is added as an inducer during the culture process of step (4), the final concentration of the added salicylic acid in the culture medium is 50 to 200 μM / L. In some embodiments, when salicylic acid is added as an inducer during the culture process of step (4), the final concentration of the added salicylic acid in the culture medium is 50 to 75 μM / L, 75 to 100 μM / L, 100 to 150 μM / L, 150 to 175 μM / L or 175 to 200 μM / L. In some embodiments, when salicylic acid is added as an inducer during the culture process of step (4), the final concentration of the added salicylic acid in the culture medium is 100 μM / L.

[0103] In some embodiments, when the inducer is added in step (4), the inducer is added in the last 5-15 days of culture. In some embodiments, when the inducer is added in step (4), the inducer is added in the last 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days of culture. In some embodiments, when the inducer is added in step (4), the inducer is added in the last 7 days of culture.

[0104] In some embodiments, in step (4), the ratio between the weight of the inoculated suspension cultured stem cells and the volume of the culture medium is 3:50 g / mL to 1:75 g / mL. In some embodiments, in step (4), the ratio between the weight of the inoculated suspension cultured stem cells and the volume of the culture medium is 3:50 g / mL to 1:25 g / mL, 1:25 g / mL to 3:100 g / mL, 3:100 g / mL to 1:50 g / mL, or 1:50 g / mL to 1:100 g / mL. In some embodiments, in step (4), the ratio between the weight of the inoculated suspension cultured stem cells and the volume of the culture medium is 1:50 g / mL.

[0105] In some embodiments, the amount of the suspended cultured Rhodiola rosea stem cells inoculated in step (4) is 1 to 8 g. In some embodiments, the amount of the Rhodiola rosea stem cells inoculated in step (4) is 1 to 2 g, 2 to 3 g, 3 to 4 g, 4 to 5 g, 5 to 6 g, 6 to 7 g or 7 to 8 g. In some embodiments, the amount of the Rhodiola rosea stem cells inoculated in step (4) is 4 g.

[0106] In some embodiments, the volume of the culture medium used in step (4) is 50-400 mL. In some embodiments, the volume of the culture medium used in step (4) is 50-100 mL, 100-150 mL, 150-200 mL, 200-250 mL, 250-300 mL, 300-350 mL or 350-400 mL. In some embodiments, the volume of the culture medium used in step (4) is 200 mL.

[0107] In some embodiments, the solid culture medium used in step (4) is the same as the solid culture medium used in step (3). In some embodiments, the solid culture medium used in step (4) is defined as the solid culture medium used in step (3).

[0108] About step (5)

[0109] In some embodiments, in step (5), the stem cells obtained in step (2) are transferred to a solid culture medium and cultured under conditions selected from a dark room, LED lighting, fluorescent lighting, and a combination thereof, preferably under LED lighting.

[0110] In some embodiments, the illumination intensity of the LED illumination in step (5) is 2000 to 6000 lux. In some embodiments, the illumination intensity of the LED illumination in step (5) is 2000 to 2200 lux, 2200 to 2400 lux, 2400 to 2600 lux, 2600 to 2800 lux, 2800 to 3000 lux, 3000 to 3200 lux, 3200 to 3400 lux, 3400 to 3600 lux, 3600 to 3800 lux, In some embodiments, the illumination intensity of the LED illumination in step (4) is 3400 to 5000 lux.

[0111] In some embodiments, in step (5), the LED illumination or the fluorescent illumination is optionally accompanied by irradiation of a 5000 to 8000K LED lamp as an external light source. In some embodiments, in step (4), the LED illumination or the fluorescent illumination is optionally accompanied by irradiation of a 5000 to 5500K, 5500 to 6000K, 6000 to 6500K, 6500 to 7000K, 7000 to 7500K or 7500 to 8000K LED lamp as an external light source.

[0112] In some embodiments, in step (5), the LED illumination or the fluorescent illumination is not accompanied by illumination with a 5000 to 8000K LED lamp as external light.

[0113] In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 8-16 hours / day. In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 8-9 hours / day, 9-10 hours / day, 10-11 hours / day, 11-12 hours / day, 12-13 hours / day, 13-14 hours / day, 14-15 hours / day or 15-16 hours / day. In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 11-13 hours / day. In some embodiments, the LED illumination or the fluorescent illumination irradiation time is 12 hours / day.

[0114] In some embodiments, in step (5), darkroom culture, LED light culture and / or fluorescence culture are performed alternately. In some embodiments, in step (4), stem cell culture is performed simultaneously under LED light and fluorescence light.

[0115] In some embodiments, the culture time in step (5) is 21-42 days. In some embodiments, the culture time in step (5) is 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days or 42 days. In some embodiments, the culture time in step (5) is 23-31 days. In some embodiments, the culture time in step (5) is 28 days.

[0116] In some embodiments, optionally, an inducer is added during the culturing process of step (5). In some embodiments, the inducer is selected from methyl jasmonate, yeast elicitor, salicylic acid or a combination thereof.

[0117] In some embodiments, when methyl jasmonate is added as an inducer during the culture process of step (5), the final concentration of the added methyl jasmonate in the culture medium is 100 to 300 μM / L. In some embodiments, when methyl jasmonate is added as an inducer during the culture process of step (5), the final concentration of the added methyl jasmonate in the culture medium is 100 to 150 μM / L, 150 to 200 μM / L, 200 to 250 μM / L, or 250 to 300 μM / L. In some embodiments, when methyl jasmonate is added as an inducer during the culture process of step (4), the final concentration of the added methyl jasmonate in the culture medium is 200 μM / L.

[0118] In some embodiments, when a yeast elicitor is added as an inducer during the culture process of step (5), the final concentration of the added yeast elicitor in the culture medium is 0.5 to 2.0 g / L. In some embodiments, when a yeast elicitor is added as an inducer during the culture process of step (5), the final concentration of the added yeast elicitor in the culture medium is 0.5 to 0.75 g / L, 0.75 to 1.0 g / L, 1.0 to 1.25 g / L, 1.25 to 1.5 g / L, 1.5 to 1.75 g / L or 1.75 to 2.0 g / L. In some embodiments, when a yeast elicitor is added as an inducer during the culture process of step (5), the final concentration of methyl jasmonate in the culture medium is 1.25 g / L.

[0119] In some embodiments, when salicylic acid is added as an inducer during the culture process of step (5), the final concentration of the added salicylic acid in the culture medium is 50 to 200 μM / L. In some embodiments, when salicylic acid is added as an inducer during the culture process of step (5), the final concentration of the added salicylic acid in the culture medium is 50 to 75 μM / L, 75 to 100 μM / L, 100 to 150 μM / L, 150 to 175 μM / L or 175 to 200 μM / L. In some embodiments, when salicylic acid is added as an inducer during the culture process of step (5), the final concentration of the added salicylic acid in the culture medium is 100 μM / L.

[0120] In some embodiments, when the inducer is added in step (5), the inducer is added in the last 5-15 days of culture. In some embodiments, when the inducer is added in step (5), the inducer is added in the last 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days of culture. In some embodiments, when the inducer is added in step (5), the inducer is added in the last 7 days of culture.

[0121] In some embodiments, in step (5), the ratio between the weight of the inoculated Rhodiola rosea stem cells and the volume of the culture medium is 1:10g / mL to 1:50g / mL. In some embodiments, in step (5), the ratio between the weight of the inoculated Rhodiola rosea stem cells and the volume of the culture medium is 1:10g / mL to 1:15g / mL, 1:15g / mL to 3:50g / mL, 3:50g / mL to 1:20g / mL, 1:20g / mL to 1:25g / mL, 1:25g / mL to 1:30g / mL, 1:30g / mL to 1:35g / mL, 1:35g / mL to 1:40g / mL, 1:40g / mL to 1:45g / mL or 1:45g / mL to 1:50g / mL. In some embodiments, in step (5), the ratio between the weight of the inoculated Rhodiola rosea stem cells and the volume of the culture medium is 3:50g / mL.

[0122] In some embodiments, the amount of Rhodiola rosea stem cells inoculated in step (5) is 1 to 8 g. In some embodiments, the amount of Rhodiola rosea stem cells inoculated in step (5) is 1 to 2 g, 2 to 3 g, 3 to 4 g, 4 to 5 g, 5 to 6 g, 6 to 7 g or 7 to 8 g. In some embodiments, the amount of Rhodiola rosea stem cells inoculated in step (5) is 6 g.

[0123] In some embodiments, the volume of the culture medium used in step (5) is 50-400 mL. In some embodiments, the volume of the culture medium used in step (5) is 50-100 mL, 100-150 mL, 150-200 mL, 200-250 mL, 250-300 mL, 300-350 mL or 350-400 mL. In some embodiments, the volume of the culture medium used in step (5) is 100 mL.

[0124] In some embodiments, the solid culture medium used in step (5) is the same as the solid culture medium used in step (2). In some embodiments, the solid culture medium used in step (5) is defined as the solid culture medium used in step (2).

[0125] Preparation method of rhodiola rosea stem cell extract

[0126] The third aspect of the present invention relates to a method for preparing a Rhodiola rosea stem cell extract, which comprises extracting Rhodiola rosea stem cells cultured according to the culture method described in the first aspect or the second aspect of the present invention.

[0127] In some embodiments, the extracting comprises the following steps:

[0128] (a) freeze-drying the Rhodiola rosea stem cells cultured by the culture method according to the first aspect or the second aspect of the present invention;

[0129] (b) extracting the freeze-dried Rhodiola rosea stem cells with a solvent selected from methanol, ethanol, polyols, ethyl acetate, water, or a mixture of one or more thereof;

[0130] (c) filtering the solution after extraction in step (2), concentrating and drying the obtained filtrate to obtain the Rhodiola rosea stem cell extract.

[0131] In some embodiments, the polyol includes propylene glycol, butylene glycol, and the like.

[0132] In some embodiments, the extraction includes extracting extracts from Rhodiola rosea stem cells according to any known method in the prior art. In some embodiments, the extraction includes ultrasonic extraction, microwave extraction, low-temperature microfluidization extraction, etc.

[0133] In some embodiments, the extraction in step (b) is ultrasonic extraction. In some embodiments, the extraction in step (b) is microwave extraction. In some embodiments, the extraction in step (b) is low-temperature microfluidic extraction.

[0134] In some embodiments, the extraction temperature in step (b) is 20-50° C. In some embodiments, the extraction temperature in step (b) is 20-25° C., 25-30° C., 30-35° C., 35-40° C., 40-45° C. or 45-50° C.

[0135] In some embodiments, the extraction in step (b) is an extraction under ultrasound. In some embodiments, the extraction in step (b) is an extraction under ultrasound, and the ultrasound frequency is 20-60KHz. In some embodiments, the extraction in step (b) is an extraction under ultrasound, and the ultrasound frequency is 20-25KHz, 25-30KHz, 30-35KHz, 35-40KHz, 40-45KHz, 45-50KHz, 50-55KHz or 55-60KHz.

[0136] In some embodiments, the extraction time of the extraction in step (b) is 0.5-5h. In some embodiments, the extraction time of the extraction in step (b) is 0.5-1h, 1-1.5h, 1.5-2h, 2-2.5h, 2.5-3h, 3-3.5h, 3.5-4h, 4-4.5h or 4.5-5h.

[0137] Use of rhodiola rosea stem cell extract for preparing medicine or cosmetics

[0138] The fifth aspect of the present invention relates to use of the Rhodiola rosea stem cell extract according to the fourth aspect of the present invention in the preparation of medicines or cosmetics.

[0139] In some embodiments, the medicine or cosmetic has whitening and / or anti-oxidation effects.

[0140] Medicines and cosmetics

[0141] The sixth aspect of the present invention relates to a medicine or cosmetic comprising the Rhodiola rosea stem cell extract according to the fourth aspect of the present invention.

[0142] In some embodiments, the composition accounts for 0.01%-20% by weight of the cosmetic or drug. In some embodiments, the composition accounts for 0.01%-0.02%, 0.02%-0.03%, 0.03%-0.04%, 0.04%-0.05%, 0.05%-0.06%, 0.06%-0.07%, 0.07%-0.08%, 0.08%-0.09%, 0.09%-0.1%, 0.1 %-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-11%, 11%-12%, 12%-13%, 13%-14%, 14%-15%, 15%-16%, 16%-17%, 17%-18%, 18%-19% or 19%-20%.

[0143] In some embodiments, the medicine or cosmetics are used for skin care. In some embodiments, the medicine and cosmetics are used for skin anti-oxidation and / or whitening. In some embodiments, the medicine and cosmetics comprise the composition according to any one of the first to fourth aspects of the present invention and one or more pharmaceutically or cosmetically acceptable carriers, diluents or excipients. In some embodiments, the medicine and cosmetics are creams, emulsions, pastes, ointments, facial masks, gels, lotions or essences. In some embodiments, the medicine and cosmetics of the present invention may also contain one or more other ingredients, such as plant extracts, nutritional additives, surfactants, flavors and fragrances, pigments, preservatives, antioxidants, moisturizers, ultraviolet absorbers, astringents, penetration aids, pH regulators, etc. Those skilled in the art can make a selection according to their common sense and specific needs.

[0144] In some embodiments, the medicine or cosmetic further comprises one or more other cosmetic active ingredients, such as but not limited to moisturizers, keratin repair agents, oil control agents, oil repellent agents, hydrating agents, anti-allergic agents, anti-aging and anti-wrinkle agents, etc. DETAILED DESCRIPTION

[0145] The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

[0146] The instruments and equipment used in the embodiments of the present invention are all conventional instruments and equipment in the field and can be replaced by instruments and equipment that meet the corresponding standards.

[0147] The Chinese herbal medicine raw materials used in the embodiments of the present invention are all commercially available unless otherwise specified, and the reagents used in the embodiments of the present invention are all commercially available analytically pure chemical reagents unless otherwise specified.

[0148] The raw materials and instruments and equipment mentioned in the present invention are all commonly used raw materials and instruments and equipment in the field, which are only examples and are not intended to limit the scope of protection of the present invention. Those skilled in the art can select equivalent raw materials and related instruments and equipment based on the disclosure of the present invention.

[0149] Experimental materials, equipment and statistical analysis methods

[0150] Experimental reagents and equipment: MS culture medium (purchased from Beijing Xinxing Zhonghe Biotechnology Co., Ltd.), improved White solid culture medium (purchased from Beijing Xinxing Zhonghe Biotechnology Co., Ltd.), fluorescent lamp (Philips, model: three-primary color 6500K), LED lamp (Guangyi plant growth lamp, model: full spectrum 30 watts), shaker (Shanghai Tiancheng, model: TS-3112S), balance (Mettler, model: PL2002), pH meter (Sartorius, model: PB-10), clean bench (Shanghai Dingke, model: SW-CJ-2G), reverse osmosis water machine (Novartis Qingyuan, model: 800g)...

[0151] Statistical methods: The experimental results are the mean ± standard deviation of multiple parallel experimental data. Paired t-test was used to test whether the differences between groups were statistically significant. The statistical software was GraphPad 5.0.

[0152] Example

[0153] Example 1: Seed germination and seedling growth

[0154] (1) Pour the seeds of Rhodiola rosea into a 2 ml centrifuge tube, treat with 75% ethanol for 20 to 60 seconds, and then remove the ethanol.

[0155] (2) Add 10% sodium hypochlorite and shake repeatedly for 3 to 10 minutes.

[0156] (3) Absorb the sodium hypochlorite and rinse with sterile water 3 to 6 times to obtain sterile seeds.

[0157] (4) Use toothpicks to neatly arrange the sterile seeds on the MS solid culture medium.

[0158] (5) Illuminate with trichromatic fluorescent light (3000-4000 lux) for 10 hours per day and culture at a temperature of 24°C ± 1°C for 1 month.

[0159] (6) Select seedlings with 2-3 leaves, transfer them to modified White solid culture medium, and switch to full-spectrum LED lighting.

[0160] (7) Full spectrum LED light (6000-7000 lux) for 12 h / day, culture at 24°C ± 1°C for 2-3 months.

[0161] For seed germination and seedling growth during the experiment, see Figure 1 .

[0162] Example 2: Induction and screening of stem cells in different parts of seedlings

[0163] (1) Select the seedlings with normal growth and well-developed root system obtained according to the steps in Example 1, separate the shoot tips under a dissecting microscope, and transfer them to MS solid medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 5.5 g / L agar powder and 30 g / L sucrose (see Figure 2 ).

[0164] (2) Cut the leaves into small pieces of about 1-2 cm, and the stems into small sections of 1-2 cm, and transfer them to MS solid medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 5.5 g / L agar powder, and 30 g / L sucrose (see Figure 2 ).

[0165] (3) The entire root system was separated from the plant. Under a dissecting microscope, the root tip was separated and the rest was cut into 1-2 cm segments and transferred to MS solid medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 5.5 g / L agar powder and 30 g / L sucrose (see Figure 2 ).

[0166] (4) Cultivate in a dark room (temperature 24°C ± 1) for 20-30 days.

[0167] (5) After culturing for 20-30 days, the white raised part of the tissue was separated and inoculated into MS solid medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 5.5 g / L agar powder and 30 g / L sucrose.

[0168] (6) White callus cell masses are formed through screening culture for 2 to 5 months.

[0169] (7) During the screening and culture process, it was found that the ability of cells in the shoot tip, stem, and leaves to proliferate and multiply became increasingly poor, while the cells in the root and root tip were loose and could continue to proliferate and expand (see Figure 3 ), white loose and active cells from the roots were selected and inoculated into MS solid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 5.5 g / L agar powder and 30 g / L sucrose to form Rhodiola rosea stem cells with loose texture, self-renewal replication ability and differentiation potential and can be cultured stably for a long time.

[0170] (8) Culture in a dark room at 24°C ± 1 for 60-90 days (see Figure 4 ).

[0171] Example 3: Suspension culture of stem cells

[0172] (1) The root tip stem cells obtained by solid culture according to Example 2 were transferred into 70 ml of liquid culture medium in a 250 ml Erlenmeyer flask, wherein the liquid culture medium was MS liquid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid and 30 g / L sucrose, so that the cells were partially exposed to the air and the shaking speed was controlled at 70 rpm.

[0173] (2) The cells are cultured in the dark for 2-5 weeks to allow them to fully adapt to the liquid environment.

[0174] (3) Add 100 ml of MS liquid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, and 30 g / L sucrose to the flask and control the shaking speed at 120 rpm to form suspended cells.

[0175] (4) Cultivate in a dark room at 24°C ± 1 on a shaking table for 20-30 days.

[0176] Example 4: Cultivation of Rhodiola rosea stem cells under different culture conditions

[0177] Starting from the seeds of Rhodiola rosea, the culture process in Examples 1-3 is performed to obtain Rhodiola rosea root stem cells that can stably reproduce in liquid culture medium, and the obtained stem cells are transferred to MS liquid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid and 30 g / L sucrose. The Rhodiola rosea stem cells are cultured by using different culture conditions (such as different light sources, different inducers, different inoculation amounts, different culture times, different amounts of culture medium, etc.) to study the conditions suitable for culturing Rhodiola rosea stem cells that can produce the most cosmetic active ingredients.

[0178] Although the inventors have done a lot of research on exploring different culture conditions, for the sake of space, the inventors list three representative culture batches as follows. Each of the three batches was carried out simultaneously in the same wave, while different batches were not carried out at the same time.

[0179] Batch I

[0180] Sample IA: 6 g of the suspended root-derived stem cells obtained in Example 3 were inoculated into 200 mL of MS liquid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid and 30 g / L sucrose, and irradiated with a 3400 lux LED light source and exogenous light (6500K LED lamp) for 12 hours every day for 4 weeks.

[0181] Sample IB: The culture steps are basically the same as those of sample IA, except that the culture under LED light source and exogenous light is replaced by culture in a dark room.

[0182] Sample IC: The culture steps are basically the same as those of sample IA, except that the culture under LED light source is replaced by culture under fluorescent light.

[0183] Sample ID: The culture steps were basically the same as those of sample IA, except that the inducer, methyl jasmonate, was added to the culture medium in the last week of culture (to a final concentration of 200 μM / L in the culture medium).

[0184] Sample IE: The root-derived stem cells obtained in Example 2 were continuously subcultured on an MS solid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 5.5 g / L agar powder and 30 g / L sucrose. 6 g of Rhodiola rosea stem cells, which have a loose texture, self-renewal and replication capabilities and differentiation potential and can be stably cultured for a long time, were inoculated into 100 mL of a solid culture medium, and irradiated with a 3400 lux LED light source and exogenous light (6500K LED lamp) for 12 hours every day for 4 weeks.

[0185] Batch II

[0186] Sample II-A: 6 g of the root-derived stem cells obtained in suspension culture in Example 3 were inoculated into 100 mL of MS liquid medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, and 30 g / L sucrose, and cultured in a dark room for 4 weeks.

[0187] Sample II-B: The culture steps are basically the same as those of sample II-A, except that the inducer, methyl jasmonate, is added to the culture medium in the last week of culture (to a final concentration of 200 μM / L in the culture medium).

[0188] Sample II-C: The culture steps are basically the same as those of sample II-A, except that an inducer, yeast elicitor, is added to the culture medium in the last week of culture (to a final concentration of 1.25 g / L in the culture medium).

[0189] Sample II-D: The culture steps are basically the same as those of sample II-A, except that the inducer, salicylic acid, is added to the culture medium in the last week of culture (to a final concentration of 100 μM / L in the culture medium).

[0190] Sample II-E: The culture steps are basically the same as those of sample II-A, except that the darkroom culture is replaced by culture under fluorescent light.

[0191] Sample II-F: The culture steps are basically the same as those of sample II-B, except that the darkroom culture is replaced by culture under fluorescent light.

[0192] Sample II-G: The culture steps are basically the same as those of sample II-C, except that the darkroom culture is replaced by culture under fluorescent light.

[0193] Sample II-H: The culture steps are basically the same as those of sample II-D, except that the darkroom culture is replaced by culture under fluorescent light.

[0194] Batch III

[0195] Sample III-A: 4 g of the suspended root-derived stem cells obtained in Example 3 were inoculated into 200 mL of MS liquid culture medium containing 1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid and 30 g / L sucrose, and cultured for 4 weeks in the presence of a 3400 lux LED light source and exogenous light (6500K LED lamp).

[0196] Sample III-B: The cultivation steps are basically the same as those of Sample III-A, except that the 3400 lux LED light source is replaced by a 5000 lux LED light source.

[0197] Sample III-C: The culture steps are basically the same as those of sample III-A, except that there is no exogenous light.

[0198] Example 5: Preparation of Rhodiola rosea stem cell extract

[0199] The Rhodiola rosea stem cells obtained in Example 4 were freeze-dried and ultrasonically extracted with methanol at a solid-liquid ratio of 1:20, with an extraction temperature of 25° C., an ultrasonic frequency of 40 KHz, and an extraction time of 1 h. After the extraction was completed, the filtrate was filtered through a Buchner funnel, and the filtrate was concentrated under reduced pressure and dried to obtain a Rhodiola rosea stem cell extract.

[0200] Example 6: Tyrosinase inhibitory activity of stem cell extracts

[0201] 1. Experimental principle: The tyrosinase inhibition test can be used to evaluate the whitening effect of cosmetic ingredients. The stronger the inhibitory activity of a cosmetic ingredient on tyrosinase, the better its whitening effect.

[0202] 2. Experimental materials: tyrosinase (Sigma, T3824-25KU), L-DOPA (Sigma, D9628), NaH2PO4 (Greagent, G81292A), Na2HPO4 (Greagent, G81291A), hydrochloric acid (CP, 36.0-38.0%, Shanghai test), kojic acid (RG, Adamas).

[0203] 3. Experimental Methods

[0204] 3.1. Solution preparation

[0205] 80 mM phosphate buffer (PB): 1) 9.60 g NaH2PO4 was dissolved in 1 L deionized water; 2) 11.36 g Na2HPO4 was dissolved in 1 L deionized water; 3) 2) was added to 1) until the pH was 6.8.

[0206] 1M HCl: Add 1 mL of concentrated HCl to 11 mL of deionized water.

[0207] 6.0 mM L-DOPA solution: Dissolve 7.1 mg of L-DOPA in 100 μL of 1 M hydrochloric acid, and add 80 mM PB to make up to 6 mL.

[0208] 250U / mL tyrosinase: Dissolve 25KU tyrosinase solid in 1mL 80mM PB, aliquot 40μL per tube and freeze at -80℃. Add PB to 4mL before use.

[0209] Kojic acid solution (positive control): Weigh 4.5 mg of kojic acid and dissolve it in 1 mL of deionized water (32 mM). Dilute it with deionized water to 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 mM for later use.

[0210] Experimental procedures

[0211] Add reagents according to Table 1 below and react, and measure the absorbance at 475nm with an enzyme reader

[0212] Table 1

[0213]

[0214] The tyrosinase inhibition rate of the sample was calculated by the following formula:

[0215] Tyrosinase inhibition rate % = [1-(A s -A sb ) / (A c -A cb )]×100%

[0216] The tyrosinase inhibition rate of samples at different concentrations was tested, and the curve was fitted by nonlinear regression using GraphPad Prism software to calculate the IC of tyrosinase inhibition. 50 value.

[0217] 4. Experimental Results

[0218] The tyrosinase inhibition IC values ​​of samples IA to IE from batch I were tested and calculated as described above. 50 The results are shown in Table 2:

[0219] Table 2

[0220] sample variable <![CDATA[Tyrosinase inhibition IC 50 (mg / mL)]]> Sample IA Liquid medium + LED + 4 weeks 1.17 Sample IB Liquid medium + dark room + 4 weeks 12.73 Sample IC Liquid medium + fluorescence + 4 weeks 11.83 Sample ID Liquid medium + LED + methyl jasmonate + 4 weeks 0.76 Sample IE Solid medium + LED + 6 weeks 4.80

[0221] From the tyrosinase inhibition IC of samples IA, IB and IC in Table 2 50 The results of the values ​​show that compared with culture in a dark room and culture under fluorescent light, Rhodiola rosea stem cells cultured under LED light contain more whitening ingredients, and the whitening activity of their extracts is significantly stronger.

[0222] From the tyrosinase inhibition IC of samples IA and ID in Table 2 50 The results of the values ​​show that after adding the inducer methyl jasmonate, Rhodiola rosea stem cells contain more whitening components and their extracts have stronger whitening activity.

[0223] From the tyrosinase inhibition IC values ​​of samples IA and IE in Table 2 50 The results of the values ​​show that compared with those cultured in solid culture medium, Rhodiola rosea stem cells cultured in liquid culture medium contain more whitening components and their extracts have stronger whitening activity.

[0224] Example 7: DPPH scavenging activity of stem cell extracts

[0225] 1. Experimental principle: DPPH is a stable free radical with a maximum absorption wavelength at 517nm. It is scavenged after accepting electrons and protons, so the absorbance at 517nm decreases. The oxidative action of free radicals can damage cells and tissues and cause pathological changes. This method predicts the antioxidant effect of the product based on the scavenging effect of the product on DPPH.

[0226] 2. Experimental materials: DPPH, epigallocatechin gallate (EGCG), vitamin C (VC), and deionized water.

[0227] 3. Experimental Methods

[0228] 3.1. Preparation of DPPH working solution

[0229] Take an appropriate amount of DPPH solid and dissolve it in anhydrous ethanol until the absorbance at 517 nm is 1.6±0.01 (take 200 μL and detect in a 96-well plate).

[0230] 3.2. Positive Control

[0231] EGCG (458.372 g / mol): Weigh 18 mg of EGCG and dissolve it in 1 mL of deionized water. Take 100 μL to 900 μL of deionized water and dilute it 1000 times to obtain a 40 μM EGCG solution. Deionized water is then diluted to 30, 20, 10, 5, 2.5, and 1.25 μM.

[0232] VC (176.13 g / mol): Weigh 14 mg VC and dissolve it in 0.5 mL deionized water. Take 100 μL to 900 μL deionized water and dilute it 1000 times to obtain a 160 μM VC solution. Deionized water is then diluted to 120, 80, 40, 20, 10, and 5 μM.

[0233] Experimental procedures

[0234] Add reagents according to Table 3 below, mix well and react for 30 min, and detect the absorbance at 517 nm with an ELISA reader.

[0235] Table 3

[0236] <![CDATA[Sample Group A s > <![CDATA[Blank group A sb > <![CDATA[Control group A c > <![CDATA[Blank Group A cb > sample 100μL 100μL — — Ethanol — 100μL — 100μL Sample solvent — — 100μL 100μL DPPH solution 100μL — 100μL —

[0237] The calculation formula is as follows:

[0238] DPPH clearance rate % = [1-(A s -A sb ) / (A c -A cb )]×100%

[0239] The DPPH scavenging rate of samples at different concentrations was tested, and the DPPH scavenging IC was calculated by curve fitting using nonlinear regression using GraphPad Prism software. 50 value.

[0240] 4. Experimental Results

[0241] The DPPH inhibition IC of samples IA to IE from batch I was tested and calculated according to the above method. 50 The results are shown in Table 4:

[0242] Table 4

[0243]

[0244]

[0245] DPPH removal of IC by samples IA, IB and IC in Table 4 50 The results of the values ​​show that compared with darkroom culture and culture under fluorescent light, Rhodiola rosea stem cells cultured under LED light contain more antioxidant components, and the antioxidant activity of their extracts is significantly stronger.

[0246] IC scavenged by DPPH of samples IA and ID in Table 4 50 DPPH Scavenging IC 50 The results of the values ​​show that compared with the culture with the inducer methyl jasmonate, the Rhodiola rosea stem cells obtained without adding methyl jasmonate contain more antioxidant components and the antioxidant activity of their extracts is stronger.

[0247] The DPPH removal IC of samples IA and IE in Table 4 50 The results of the values ​​show that compared with those cultured in solid culture medium, Rhodiola rosea stem cells cultured in liquid culture medium contain more antioxidant components and their extracts have stronger antioxidant activity.

[0248] The DPPH scavenging IC values ​​of batch II samples II-A to II-H were tested and calculated according to the above method. 50 The results are shown in Table 5:

[0249] Table 5

[0250] sample variable <![CDATA[DPPH scavenging IC 50 (mg / mL)]]> Sample II-A Dark room, no inducer 0.759 Sample II-B Darkroom + Methyl Jasmonate 0.292 Sample II-C Darkroom + yeast elicitor 0.322 Sample II-D Darkroom + Salicylic Acid 0.352 Sample II-E Fluorescent light, no inducers 0.434 Sample II-F Fluorescent light irradiation + methyl jasmonate 0.537 Sample II-G Fluorescent light irradiation + yeast elicitor 0.375 Sample II-H Fluorescent light + salicylic acid 0.375

[0251] The DPPH scavenging IC values ​​of batch III samples III-A to III-C were tested and calculated according to the above method. 50 The results are shown in Table 6:

[0252] Table 6

[0253]

[0254]

Claims

1. A method for culturing Rhodiola rosea stem cells, the method comprising the following steps: (1) inoculating sterile Rhodiola rosea seeds and culturing them until they germinate and grow into seedlings; (2) isolating the roots or root tips of the seedlings in step (1), culturing Rhodiola rosea stem cells on a solid culture medium, and further amplifying the cultured Rhodiola rosea stem cells to obtain stem cell aggregates; (3) transferring the stem cell mass formed in step (2) to a liquid culture medium for suspension culture for a period of time until the stem cells are fully adapted to the liquid culture conditions and can stably reproduce in the liquid culture medium; and (4) Inoculating the stem cells cultured in suspension in step (3) into a liquid culture medium, and culturing under conditions selected from a dark room, LED illumination, fluorescent illumination, and a combination thereof, preferably culturing under LED illumination.

2. A method for culturing Rhodiola rosea stem cells, the method comprising the following steps: (1) inoculating sterile Rhodiola rosea seeds and culturing them until they germinate and grow into seedlings; (2) isolating the roots or root tips of the seedlings in step (1), culturing Rhodiola rosea stem cells on a solid culture medium, and further amplifying the cultured Rhodiola rosea stem cells to obtain stem cell aggregates; and (5) Transferring the stem cells obtained in step (2) to a solid culture medium, and culturing them under conditions selected from a dark room, LED illumination, fluorescent illumination, and a combination thereof, preferably culturing them under LED illumination.

3. The method for culturing Rhodiola rosea stem cells according to claim 1 or 2, wherein in step (1), the aseptically treated seeds are first inoculated in MS medium and cultured under 8-16 hours / day of trichromatic fluorescent light until the seeds germinate; and then the germinated seeds are transferred to a modified White solid medium and cultured under 8-16 hours / day of full-spectrum LED light until seedlings grow.

4. The method for culturing Rhodiola rosea stem cells according to any one of claims 1 to 3, wherein in step (2), the roots or root tips of the seedlings separated in step (1) are cultured in the solid culture medium in step (2) until white protuberance-like Rhodiola rosea stem cells are produced on the tissue, and then the white protuberance-like Rhodiola rosea stem cells are separated and inoculated separately into another solid culture medium of the same composition for amplification culture, and after multiple generations of amplification culture, white loose stem cell clusters are obtained.

5. The method for culturing Rhodiola rosea stem cells according to any one of claims 1 and 3-4, wherein in step (3), the stem cell agglomerates formed in step (2) are first transferred to a liquid culture medium of volume A and suspended and cultured at a rotation speed of M for a period of time until no browning occurs, and then transferred to a liquid culture medium of volume B and suspended and cultured at a rotation speed of N for a period of time, wherein the volume A is smaller than the volume B, and the rotation speed M is smaller than the rotation speed N.

6. The method for culturing Rhodiola rosea stem cells according to any one of claims 1 and 3-5, wherein in step (4), the culture time is 23-31 days, the ratio between the weight of the inoculated suspension cultured stem cells and the volume of the culture medium is 3:50 g / mL to 1:75 g / mL, and optionally, an inducer is added during the culture process.

7. The method for culturing Rhodiola rosea stem cells according to any one of claims 2 and 3-5, wherein in step (5), the culture time is 23-31 days, the ratio between the weight of the inoculated suspension cultured stem cells and the volume of the culture medium is 1:10 g / mL to 1:50 g / mL, preferably 3:50 g / mL, and optionally, an inducer is added during the culture process.

8. The method for culturing Rhodiola rosea stem cells according to claim 6 or 7, wherein the inducer is selected from methyl jasmonate, yeast elicitor, salicylic acid or a combination thereof.

9. The method for culturing Rhodiola rosea stem cells according to any one of the preceding claims, wherein the culturing in steps (2) and (3) is performed in a dark room.

10. The method for culturing Rhodiola rosea stem cells according to any one of the preceding claims, wherein the solid culture medium used in steps (2) and (5) is an MS culture medium containing 0.5-2.0 mg / L of 6-benzylaminopurine and 0.1-1.0 mg / L of naphthaleneacetic acid, and the liquid culture medium used in steps (3) and (4) is an MS culture medium containing 0.5-2.0 mg / L of 6-benzylaminopurine and 0.1-1.0 mg / L of naphthaleneacetic acid and not containing agar.

11. The method for culturing Rhodiola rosea stem cells according to any one of the preceding claims, wherein the Rhodiola rosea is selected from the group consisting of Rhodiola angusta, Rhodiola rosea, Rhodiola sacra, Rhodiola calliantha (H. Ohba) H. Ohba, Rhodiola cretinii (Raym.-Hamet) H. Ohba, Rhodiola heterodonta (Hook.f. & Thomson) Boriss., Rhodiola kirilowii (Regel) Maxim., Rhodiola linearifolia A. Bor., Rhodiola kirilowii (Regel) Maxim., Rhodiola rosea L., Rhodiola serrata The invention relates to Rhodiola rosea. The invention relates to Rhodiola rosea var. H.Ohba), Rhodiola dumulosa (Franch.) SHFu), Rhodiola angusta, Rhodiola fastigiate, Rhodiola quadrifida, Rhodiola sachalinensis, Rhodiola tangutica, Rhodiola wallichiana, Rhodiola yunnanensis and Rhodiola himalensis, and Rhodiola grandiflora is preferred.

12. A method for preparing a Rhodiola rosea stem cell extract, the method comprising extracting the Rhodiola rosea stem cells cultured according to the culture method according to any one of claims 1 to 11.

13. The method according to claim 12, wherein the extracting comprises the following steps: (a) freeze-drying the Rhodiola rosea stem cells cultured according to any one of the culture methods of claims 1 to 11; (b) extracting the freeze-dried Rhodiola rosea stem cells with a solvent selected from methanol, ethanol, polyols, ethyl acetate, water, or a mixture of one or more thereof; (c) filtering the solution after extraction in step (2), concentrating and drying the obtained filtrate to obtain the Rhodiola rosea stem cell extract.

14. A Rhodiola rosea stem cell extract, the extract being prepared by the method according to claim 12 or 13.

15. Use of the Rhodiola rosea stem cell extract according to claim 14 in the preparation of medicines or cosmetics.

16. The use according to claim 15, wherein the medicine or cosmetic has whitening and / or anti-oxidation effects.

17. A medicine or cosmetic comprising the Rhodiola rosea stem cell extract according to claim 14.

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