Preparation method and use of Centella asiatica stem cells with high production of madecassoside

Abstract

The present invention provides a preparation method and use of Centella asiatica stem cells with high madecassoside production, belonging to the technical field of stem cells. The preparation method includes disinfection, induction, proliferation, and cultivation of Centella asiatica stem cells with high madecassoside production, etc. The method of the present invention has unexpected technical effects such as low contamination rate, high induction rate, high proliferation multiple, and high madecassoside content in the obtained Centella asiatica stem cells with high madecassoside production, which is conducive to industrial production and can be used as a skin care ingredient in products with anti-inflammatory, soothing and other effects.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a preparation method and application of Centella asiatica stem cells with high production of madecassoside. Background Art

[0002] Centella asiatica (L.) Urban. is a perennial creeping herb of the genus Centella in the family Apiaceae. It grows in humid areas, such as country roadsides, rivers and ditches. It is widely distributed in tropical and subtropical regions of the world. Its distribution in my country is mainly concentrated in various areas south of the Yangtze River. Centella asiatica is a commonly used Chinese medicine included in the Chinese Pharmacopoeia and has been used in folk medicine for hundreds of years. It is cold in nature, bitter and pungent in taste, and has the effects of clearing away heat and dampness, as well as reducing swelling and detoxifying. It is suitable for symptoms such as damp heat, diarrhea, hematuria and sores. It is also used clinically to treat injuries and skin diseases. Its mechanism of action includes promoting fibroblast proliferation, increasing collagen synthesis and the content of intracellular fibronectin, improving the tensile strength of new skin, and inhibiting the inflammatory phase of hypertrophic scars and keloids.

[0003] The results of experimental research show that the active compounds of Centella asiatica include pentacyclic triterpenes, asiaticoside, madecassoside, asiatic acid, madecassoside, etc. Among them, madecassoside is the main active ingredient in Centella asiatica, which has the effects of promoting wound healing, repairing skin scars, anti-allergy, anti-inflammatory, whitening, anti-oxidation, anti-gastric ulcer and treating hepatitis. Centella asiatica has been widely used in medicine, health food and beauty, and has broad market prospects. However, 90% of the raw materials of Centella asiatica in the world rely on wild resources. Due to the process of urbanization and the use of herbicides, Centella asiatica is rarely seen around human settlements and farmland and mountains. Therefore, introduction and cultivation, artificial breeding, etc. have become new ways to effectively utilize Centella asiatica resources.

[0004] However, physiologically active substances are difficult to develop industrially for the following main reasons: the content of physiologically active substances in plants is very limited; the growth rate of plants is very slow; physiologically active substances derived from plants are only present in small amounts in specific organs of plants; environmental problems related to natural destruction are involved; the chemical structure of physiologically active substances derived from plants is very complex and requires a multi-step polymerization process, resulting in economic problems of high production costs. Due to these reasons, it is very difficult to stably supply physiologically active substances derived from plants for commercialization.

[0005] Some scholars have conducted a comprehensive evaluation of the quality of 29 batches of Centella asiatica from different origins and found that the content of madecassoside in wild Centella asiatica is 0.541% to 1.915% (see "Liu Fusong, Li Yaoyao, Nie Xuqiang, et al. Evaluation of Centella asiatica quality based on sensory characteristics combined with grey correlation method [J]. Chinese Journal of New Drugs, 2021, 30(20): 1881-1889."). Therefore, the content of madecassoside in wild Centella asiatica is low and difficult to apply industrially.

[0006] Plants are an important source for producing a large number of natural products. The common strategy for obtaining natural products is to extract them from the original plants, but the resources and growth rate of plants usually limit the yield of natural products. As one of the bioengineering technologies, the plant cell culture method has long been evaluated as the most ideal technology that can provide useful substances from plants without causing environmental problems. Plant stem cell culture technology has been introduced for the mass production of many useful ingredients. A variety of plant-derived compounds are used in various fields such as medicines, foods and cosmetics. The production of useful substances through plant stem cell culture technology has many advantages over the method of directly extracting useful substances from plants. In particular, unlike existing extraction methods, the plant stem cell culture method is considered to be the best method that allows continuous production without being affected by the external environment, thereby solving prominent problems such as ecosystem destruction. Plant stem cells also have the characteristics of short growth cycle and high and stable content of target secondary metabolites.

[0007] Patent application CN112746054A discloses a method for culturing Centella asiatica stem cells, but this method has problems such as the introduction of copper sulfate solution, low stem cell proliferation multiples, and low madecassoside content in the obtained Centella asiatica stem cells.

[0008] Therefore, there is still an urgent need for a Centella asiatica stem cell with a high stem cell proliferation multiple and a high madecassoside content and a culture method thereof. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a method for isolating and culturing Centella asiatica stem cell lines.

[0011] A method for isolating and culturing a Centella asiatica stem cell line, comprising the following steps:

[0012] (1) Selection and disinfection of explants: Take the stem tip of a Centella asiatica seedling, disinfect it, and obtain an explant;

[0013] (2) Isolation and induction of meristem: The shoot tip containing meristem in the explant sterilized in step (1) is stripped and placed in an induction medium for induction culture to obtain induced Centella asiatica stem cells; the induction medium comprises B5 medium, based on the volume of the B5 medium, 30 g / L of sucrose (i.e., 30 g of sucrose is added to every 1 L of B5 medium, and the definitions of other similar descriptions in this article are the same), 6 g / L of agar, 1.5 mg / L-2.0 mg / L of IAA (indoleacetic acid) and 0.5 mg / L-1 mg / L of TDZ (thidiazuron), and the pH of the induction medium is 5.8-6.2;

[0014] (3) Proliferation culture: The induced Centella asiatica stem cells obtained in step (2) are transferred to a proliferation medium for culture to obtain the Centella asiatica stem cell line.

[0015] In some embodiments, the induction medium comprises B5 medium, based on the volume of B5 medium, 30 g / L sucrose, 6 g / L agar, 1.5 mg / L IAA (indoleacetic acid) and 0.5 mg / L TDZ (thidiazuron) are added, and the pH of the induction medium is 6.0.

[0016] In some embodiments, the pH of the induction medium is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0017] In some embodiments, the pH of the induction medium is adjusted using aqueous sodium hydroxide solution and / or hydrochloric acid or using aqueous NaOH solution and / or aqueous HCl solution.

[0018] In some embodiments, the proliferation medium in step (3) includes proliferation medium A and / or proliferation medium B;

[0019] The proliferation medium A comprises, based on the volume of the B5 medium, 20 g / L of sucrose, 10 g / L of glucose, 6 g / L of agar, 0.5 mg / L-1.0 mg / L of TDZ (thidiazuron), and 1.0 mg / L-2.0 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid), and the pH of the proliferation medium A is 5.8-6.2;

[0020] The proliferation medium B includes, on the basis of B5 medium, 20g / L sucrose, 10g / L glucose, 6g / L agar, 0.5mg / L-1.0mg / L NAA (naphthaleneacetic acid), 0.5mg / L-1.0mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5mg / L-1mg / L TDZ and 0.5-1mg / L 6-BA (6-benzylaminoadenine) are additionally added, based on the volume of B5 medium, and the pH of the proliferation medium B is 5.8-6.2.

[0021] In some embodiments, the proliferation medium A is based on the B5 medium, with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L TDZ (thidiazuron), and 1.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) added to the volume of the B5 medium, and the pH of the proliferation medium A is 6.0.

[0022] In some embodiments, the proliferation medium B is based on B5 medium, with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L NAA (naphthaleneacetic acid), 0.5 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L TDZ and 0.5 mg / L 6-BA (6-benzylaminoadenine) added to the volume of B5 medium, and the pH of the proliferation medium B is 6.0.

[0023] In some embodiments, the pH of the proliferation medium A is adjusted using aqueous NaOH solution and / or HCl or using aqueous NaOH solution and / or aqueous HCl solution.

[0024] In some embodiments, the pH of the proliferation medium B is adjusted using an aqueous NaOH solution and / or HCl solution or using an aqueous NaOH solution and / or an aqueous HCl solution.

[0025] In some embodiments, the pH of the proliferation medium A is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0026] In some embodiments, the pH of the proliferation medium B is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0027] In some preferred embodiments, the culturing in step (3) includes first culturing with proliferation medium A and then switching to proliferation medium B for culturing.

[0028] In some embodiments, the culture temperature for culturing using proliferation medium A is 25°C±2°C.

[0029] In some embodiments, the culture illumination condition for culturing using proliferation medium A is under dark conditions.

[0030] In some embodiments, the culture time for culturing using proliferation medium A is 7 days to 14 days. In some embodiments, the culture time for culturing using proliferation medium A is 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0031] In some embodiments, the culture temperature for culturing using proliferation medium B is 25°C±2°C.

[0032] In some embodiments, the culture illumination condition for culturing using proliferation medium B is under dark conditions.

[0033] In some embodiments, the culture time for culturing using proliferation medium B is 7 days to 14 days. In some embodiments, the culture time for culturing using proliferation medium B is 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days.

[0034] In some embodiments, the culture temperature of the induction culture in step (2) is 25°C±2°C.

[0035] In some embodiments, the induced culture in step (2) is carried out under dark conditions.

[0036] In some embodiments, the culturing time of the induced culture in step (2) is 7 days to 14 days. In some embodiments, the culturing time of the induced culture in step (2) is 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days.

[0037] In some embodiments, the disinfection of step (1) includes oscillating and soaking in a 1wt% NaClO aqueous solution for 4min-15min (e.g., 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min), rinsing with sterile water for 3-5 times, oscillating and soaking in 75% alcohol for 30s, rinsing with sterile water for 3-5 times, and absorbing the sterile water with sterile filter paper.

[0038] In some preferred embodiments, the disinfection in step (1) comprises immersing in a 1wt% NaClO aqueous solution with shaking for 8 minutes, rinsing with sterile water for 3-5 times, immersing in 75% alcohol with shaking for 30 seconds, rinsing with sterile water for 3-5 times, and absorbing the sterile water with sterile filter paper.

[0039] In some embodiments, the disinfection further comprises flushing with water for 30 min-40 min before using the 1 wt % NaClO aqueous solution.

[0040] In a second aspect, the present invention provides a method for culturing Centella asiatica stem cells with high production of madecassoside.

[0041] A method for culturing Centella asiatica stem cells with high madecassoside production, comprising: culturing the Centella asiatica stem cell line obtained by the method of the first aspect in a culture medium, filtering after the cultivation to obtain the Centella asiatica stem cells with high madecassoside production.

[0042] In some preferred embodiments, the culture medium is based on the B5 liquid culture medium, and 0-0.1 mmol / L of inducer, 20 g / L-30 g / L of sucrose, 0-10 g / L of glucose, 0.5-2.0 mg / L of TDZ and 1.0-2.0 mg / L of 2,4-D are additionally added, based on the volume of the B5 liquid culture medium, and the pH of the culture medium is 5.8-6.2; the inducer is SA (salicylic acid), MeJA (methyl jasmonate), gibberellin, abscisic acid or ethylene; preferably SA (salicylic acid).

[0043] In some embodiments, the culture medium is based on the B5 liquid culture medium, with 0.05 mmol / L SA, 30 g / L sucrose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D added thereto, based on the volume of the B5 liquid culture medium, and the pH of the culture medium is 6.0.

[0044] In some embodiments, the culture medium is based on the B5 liquid culture medium, with 0.05 mmol / L SA, 25 g / L sucrose, 5 g / L glucose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D added thereto, based on the volume of the B5 liquid culture medium, and the pH of the culture medium is 6.0.

[0045] In some preferred embodiments, the culture medium is based on B5 liquid culture medium, and 0.05 mmol / L SA (salicylic acid), 20 g / L sucrose, 10 g / L glucose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D are further added, and the pH of the culture medium is 6.0.

[0046] In some embodiments, the pH of the culture solution is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0047] In some embodiments, the pH of the culture solution is adjusted using an aqueous NaOH solution and / or HCl or using an aqueous NaOH solution and / or an aqueous HCl solution.

[0048] In some embodiments, the illumination condition of the culture is a light intensity of 100 μmol·m -2 ·s -1 -200 μmol·m -2 ·s -1 In some embodiments, the illumination condition of the culture is a light intensity of 100 μmol·m -2 ·s -1 , 110 μmol·m -2 ·s -1 , 120 μmol·m -2 ·s -1 、130μmol·m -2 ·s -1 , 140 μmol·m -2 ·s -1 , 150 μmol·m -2 ·s -1 , 160 μmol·m -2 ·s -1 , 170 μmol·m -2 ·s -1 , 180 μmol·m -2 ·s -1 , 190 μmol·m -2 ·s -1 or 200 μmol·m -2 ·s -1 .

[0049] In some embodiments, the photoperiod of the culture is 0h / 24h-24h / 0h. In some embodiments, the photoperiod of the culture is 12h / 12h (indicating that the light-dark alternation rule is 12h of light and 12h of darkness). In some embodiments, the photoperiod of the culture is 0h / 24h (indicating that the light-dark alternation rule is 0h of light and 24h of darkness), 6h / 18h (indicating that the light-dark alternation rule is 6h of light and 18h ​​of darkness), 12h / 12h (indicating that the light-dark alternation rule is 12h of light and 12h of darkness), 18h / 6h (indicating that the light-dark alternation rule is 18h of light and 6h of darkness) or 24h / 0h (indicating that the light-dark alternation rule is 24h of light and 0h of darkness). In some embodiments, the photoperiod of the culture is 12h / 12h (indicating that the light-dark alternation rule is 12h of light and 12h of darkness).

[0050] In some embodiments, the culture time of the culture is 4 days to 6 days. In some embodiments, the culture time of the culture is 4 days, 5 days or 6 days.

[0051] In some embodiments, the stirring speed of the culture is 50rpm-150rpm. In some embodiments, the stirring speed of the culture is 100rpm. In some embodiments, the stirring speed of the culture is 50rpm, 60rpm, 70rpm, 80rpm, 90rpm, 100rpm, 110rpm, 120rpm, 130rpm, 140rpm or 150rpm.

[0052] In some embodiments, the red-blue light ratio R:B (ratio of red light to blue light) in the culture illumination is 7:3.

[0053] In a third aspect, the present invention provides an induction culture medium for inducing Centella asiatica meristems to obtain Centella asiatica stem cells.

[0054] An induction culture medium for inducing Centella asiatica meristems to obtain Centella asiatica stem cells. The induction culture medium is prepared by adding 30 g / L of sucrose, 6 g / L of agar, 1.5 mg / L-2.0 mg / L of IAA (indoleacetic acid) and 0.5 mg / L-1 mg / L of TDZ (thidiazuron) to a B5 culture medium based on the volume of the B5 culture medium. The pH value of the induction culture medium is 5.8-6.2.

[0055] In some embodiments, the induction medium comprises B5 medium, based on the volume of B5 medium, 30 g / L sucrose, 6 g / L agar, 1.5 mg / L IAA (indoleacetic acid) and 0.5 mg / L TDZ (thidiazuron) are added, and the pH of the induction medium is 6.0.

[0056] In some embodiments, the pH of the induction medium is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0057] In some embodiments, the pH of the induction medium is adjusted using aqueous sodium hydroxide solution and / or hydrochloric acid or using aqueous NaOH solution and / or aqueous HCl solution.

[0058] In a fourth aspect, the present invention provides a proliferation culture medium for proliferating Centella asiatica stem cells.

[0059] A proliferation medium for proliferating Centella asiatica stem cells, comprising proliferation medium A or proliferation medium B;

[0060] The proliferation medium A is based on the B5 medium, and is further supplemented with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L-1.0 mg / L TDZ (thidiazuron), and 1.0 mg / L-2.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), based on the volume of the B5 medium, and the pH of the proliferation medium A is 5.8-6.2;

[0061] The proliferation medium B is based on the B5 medium, and is additionally supplemented with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L-1.0 mg / L NAA (naphthaleneacetic acid), 0.5 mg / L-1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L-1 mg / L TDZ and 0.5-1 mg / L 6-BA (6-benzylaminoadenine), based on the volume of the B5 medium. The pH of the proliferation medium B is 5.8-6.2.

[0062] In some embodiments, the proliferation medium A is based on the B5 medium, with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L TDZ (thidiazuron), and 1.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) added to the volume of the B5 medium, and the pH of the proliferation medium A is 6.0.

[0063] In some embodiments, the proliferation medium B is based on B5 medium, with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L NAA (naphthaleneacetic acid), 0.5 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L TDZ and 0.5 mg / L 6-BA (6-benzylaminoadenine) added to the volume of B5 medium, and the pH of the proliferation medium B is 6.0.

[0064] In some embodiments, the pH of the proliferation medium A is adjusted using aqueous NaOH solution and / or HCl or adjusted using aqueous NaOH solution and / or aqueous HCl solution.

[0065] In some embodiments, the pH of the proliferation medium B is adjusted using an aqueous NaOH solution and / or HCl or adjusted using an aqueous NaOH solution and / or an aqueous HCl solution.

[0066] In some embodiments, the pH of the proliferation medium A is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0067] In some embodiments, the pH of the proliferation medium B is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0068] In a fifth aspect, the present invention provides a culture medium for culturing Centella asiatica stem cells with a high madecassoside content.

[0069] A culture medium for culturing Centella asiatica stem cells with a high madecassoside content, wherein the culture medium is prepared by adding, based on the volume of the B5 liquid culture medium, 0-0.1 mmol / L of an inducer, 20 g / L-30 g / L of sucrose, 0-10 g / L of glucose, 0.5-2.0 mg / L of TDZ and 1.0-2.0 mg / L of 2,4-D, and the pH of the culture medium is 5.8-6.2; the inducer is SA (salicylic acid), MeJA (methyl jasmonate), gibberellin, abscisic acid or ethylene; preferably SA (salicylic acid).

[0070] In some embodiments, the pH of the culture solution is 5.8, 5.9, 6.0, 6.1 or 6.2.

[0071] In some embodiments, the pH of the culture solution is adjusted using an aqueous NaOH solution and / or HCl or is adjusted using an aqueous NaOH solution and / or an aqueous HCl solution.

[0072] In some embodiments, the culture medium is based on the B5 liquid culture medium, with 0.05 mmol / L SA, 30 g / L sucrose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D added thereto, based on the volume of the B5 liquid culture medium, and the pH of the culture medium is 6.0.

[0073] In some embodiments, the culture medium is based on the B5 liquid culture medium, with 0.05 mmol / L SA, 25 g / L sucrose, 5 g / L glucose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D added thereto, based on the volume of the B5 liquid culture medium, and the pH of the culture medium is 6.0.

[0074] In some preferred embodiments, the culture medium is based on B5 liquid culture medium, and 0.05 mmol / L SA (salicylic acid), 20 g / L sucrose, 10 g / L glucose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D are further added, and the pH of the culture medium is 6.0.

[0075] In a sixth aspect, the present invention provides a Centella asiatica stem cell freeze-dried powder.

[0076] A Centella asiatica stem cell freeze-dried powder, the preparation method of which comprises: freeze-drying the Centella asiatica stem cells with high madecassoside production obtained by the culturing method described in the second aspect to obtain the Centella asiatica stem cell freeze-dried powder.

[0077] In a seventh aspect, the present invention provides a Centella asiatica stem cell.

[0078] A Centella asiatica stem cell, which is selected from the Centella asiatica stem cell line obtained by culturing according to the method of the first aspect or the Centella asiatica stem cell with high production of madecassoside obtained by the culturing method of the second aspect.

[0079] In an eighth aspect, the present invention provides a use of the Centella asiatica stem cells with high production of madecassoside obtained by the culture method described in the second aspect or the Centella asiatica stem cell freeze-dried powder described in the sixth aspect.

[0080] A use of the Centella asiatica stem cells with high production of madecassoside obtained by the culture method of the second aspect or the Centella asiatica stem cell freeze-dried powder of the sixth aspect in the preparation of cosmetics with anti-inflammatory or soothing effects.

[0081] Beneficial Effects

[0082] Compared with the prior art, the technical solution provided by the present invention has at least one of the following beneficial technical effects:

[0083] (1) The optimal time for the present invention to immerse and disinfect the stem tip explants of Centella asiatica using a 1 wt % NaClO aqueous solution is 8 minutes, which is beneficial for avoiding contamination and improving the induction rate, and has an unexpected technical effect.

[0084] (2) Compared with the use of other auxins, the addition of indoleacetic acid to the induction medium has an unexpected technical effect of increasing the induction rate.

[0085] (3) Compared with the use of other cytokinins, the addition of thiadiazole to the induction medium has an unexpected technical effect of increasing the induction rate.

[0086] (4) Compared with the use of other auxins, the addition of 2,4-dichlorophenoxyacetic acid to the proliferation medium A has the unexpected technical effect of increasing the proliferation multiple and increasing the content of hydroxy-madecassoside in the high-yield hydroxy-madecassoside-producing Centella asiatica stem cells obtained by subsequent culture in the culture medium.

[0087] (5) Compared with the use of other cytokinins, the addition of thidiazuron to the proliferation medium A has the unexpected technical effect of increasing the proliferation multiple and increasing the content of madecassoside in the high-yielding Centella asiatica stem cells obtained by subsequent culture in the culture medium.

[0088] (6) Compared with the use of a single proliferation culture medium, the present invention uses proliferation culture medium A and proliferation culture medium B for alternating culture, which has an unexpected technical effect of increasing the proliferation multiple.

[0089] (7) Compared with the use of other inducers, the addition of salicylic acid to the culture medium of the present invention is more conducive to increasing the content of madecassoside in the Centella asiatica stem cells with high madecassoside production obtained after culture in the culture medium, which has an unexpected technical effect.

[0090] (8) Compared with other red-blue light ratios, the red-blue light ratio R:B=7:3 of the present invention is more conducive to increasing the content of madecassoside in the Centella asiatica stem cells with high hydroxymadecassoside production obtained after culture in the culture medium, and has unexpected technical effects.

[0091] (9) Compared with other basal culture media, the use of B5 basal culture medium is more conducive to improving the induction rate and has unexpected technical effects.

[0092] (10) Compared with the culture medium without SA, the culture medium with SA added in the present invention can greatly increase the content of hydroxymadecassoside in the cultured stem cells, which has an unexpected technical effect.

[0093] (11) The high-madecassoside-yielding Centella asiatica stem cells prepared by the present invention have a high content of madecassoside. The obtained high-madecassoside-yielding Centella asiatica stem cells have anti-inflammatory, soothing and other effects, and can be used as an ingredient in products with anti-inflammatory, soothing and other effects.

[0094] Terminology

[0095] In the above text of the present invention, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the value of each number may vary by less than ±10% or reasonable differences considered by those skilled in the art, such as ±1%, ±2%, ±3%, ±4% or ±5%.

[0096] The terms "optional", "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur.

[0097] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0098] The term "wt%" means percent by mass.

[0099] Induction rate (%) = number of explants producing stem cells / total number of inoculated explants × 100%.

[0100] Contamination rate (%) = (number of contaminated tissue blocks / total number of tissue blocks) × 100%.

[0101] The "photoperiod" mentioned in this article refers to the number of hours of alternating light and darkness in a day. For example, a photoperiod of 6h / 18h means 6h of light and 18h ​​of darkness in a day; the definitions of other photoperiod values ​​in this article are similar.

[0102] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction. DETAILED DESCRIPTION

[0103] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0104] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0105] The source of the Centella asiatica used in the present invention is: Qingdao Baicaoxiang Aromatic Plant Co., Ltd.

[0106] The B5 medium described in each embodiment of the present invention: Take B5 medium base salt (containing vitamins), add water to dissolve to a concentration of 3163.98 mg / L, and obtain B5 medium. The product composition (mg / L) of the B5 medium is:

[0107] Product composition: (mg / L)

[0108] <![CDATA[NH4SO4]]> 134 <![CDATA[MnSO4·H2O]]> 10 <![CDATA[CaCl2]]> 113.23 <![CDATA[Na2MoO4·2H2O]]> 0.25 <![CDATA[MgSO4]]> 121.56 KI 0.75 <![CDATA[KNO3]]> 2500 <![CDATA[ZnSO4·7H2O]]> 2 <![CDATA[NaH2PO4]]> 130.44 moy-Inositol 100 <![CDATA[H3BO3]]> 3 Nicotinic acid 1 <![CDATA[CoCl2·6H2O]]> 0.025 Pyridoxine HCl 1 <![CDATA[CuSO4·5H2O]]> 0.025 Thiamine HCl 10 FeNaEDTA 36.7 - - ;

[0109] The source information of the B5 culture medium salt (containing vitamins) is: Coolaber (Beijing Coolaber Technology Co., Ltd.), item number: PM1321.

[0110] The B5 liquid culture medium described in each embodiment of the present invention is the same as the B5 culture medium described in each embodiment of the present invention as described above.

[0111] Madecassoside content detection method: Use high performance liquid chromatography to determine the content of asiatica. The liquid chromatography analysis conditions are as follows:

[0112] a) Chromatographic column: ZORBAX StableBond C18 (4.6 mm x 250 mm, 5 μm);

[0113] b) Flow rate: 1.0 mL / min;

[0114] c) Mobile phase: acetonitrile: water = 80:20;

[0115] d) Elution method: isocratic elution;

[0116] e) Injection volume: 10 μL;

[0117] f) Column temperature: 25°C;

[0118] g) Wavelength: 205nm.

[0119] Madecassoside reference solution: Accurately weigh 10.0 mg of madecassoside reference into a 10 mL volumetric flask, and then dilute to the mark with methanol. This will give a standard stock solution with a mass concentration of 1.0 mg / mL, which should be stored at -20°C.

[0120] Madecassoside working solution: Use methanol to prepare the above reference solution into a series of standard working solutions with mass concentrations of 5, 10, 20, 50, and 100 μg / mL, and prepare them before use.

[0121] Test solution: Accurately weigh 1.000 g of Centella asiatica stem cell powder (passed through a 80-mesh sieve), place it in a 50-mL stoppered conical flask, accurately add 10 mL of methanol, seal the flask and weigh it, soak it for 60 min, sonicate it for 30 min, cool it and weigh it again, make up the weight loss with methanol, shake it well, filter it through a 0.45 μm microporous filter membrane, and the filtrate obtained is the test solution.

[0122] Example 1: Preparation of Centella asiatica stem cell line

[0123] The following steps were used to isolate and culture the Centella asiatica stem cell line:

[0124] (1) Selection and disinfection of explants: The stem tip of a Centella asiatica seedling is taken and disinfected to obtain an explant; the disinfection comprises: first rinsing with water for 30 minutes; then oscillating and soaking in a 1 wt % NaClO aqueous solution for 8 minutes, rinsing with sterile water 3 times, oscillating and soaking in 75% alcohol for 30 seconds, rinsing with sterile water 3 times, and absorbing the sterile water with sterile filter paper;

[0125] (2) Isolation and induction of meristem-containing tissue: The stem tip containing meristem-containing tissue in the explant sterilized in step (1) is separated, and the stem tip containing meristem-containing tissue is placed in an induction medium for induction culture at 25° C. and in the dark for 7 days to obtain induced Centella asiatica stem cells;

[0126] The induction medium includes, based on the volume of the B5 medium, 30 g / L of sucrose (i.e., 30 g of sucrose is further added to each 1 L of the B5 medium, and the definitions of other similar descriptions in this article are similar), 6 g / L of agar, 1.5 mg / L of IAA (indoleacetic acid) and 0.5 mg / L of TDZ (thidiazuron), and the pH of the induction medium is 6.0 (pH is adjusted with NaOH aqueous solution and HCl);

[0127] (3) Proliferation culture: the induced Centella asiatica stem cells obtained in step (2) are transferred to proliferation medium A for culturing at 25° C. in the dark for 10 days, and then switched to proliferation medium B for culturing at 25° C. in the dark for 10 days to obtain the Centella asiatica stem cell line;

[0128] The proliferation medium A is based on the B5 medium, and is further supplemented with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L TDZ (thidiazuron), and 1.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) based on the volume of the B5 medium. The pH of the proliferation medium A is 6.0 (the pH is adjusted with an aqueous NaOH solution and / or HCl);

[0129] The proliferation medium B is based on the B5 medium, and is further supplemented with 20 g / L sucrose, 10 g / L glucose, 6 g / L agar, 0.5 mg / L NAA (naphthaleneacetic acid), 0.5 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L TDZ and 0.5 mg / L 6-BA (6-benzylaminoadenine), based on the volume of the B5 medium. The pH of the proliferation medium B is 6.0 (the pH is adjusted with an aqueous NaOH solution and / or HCl).

[0130] In this embodiment, compared with that before induction, the proliferation multiple of the Centella asiatica stem cells obtained after step (2) and step (3) is 9.2.

[0131] Example 2: Method for culturing Centella asiatica stem cells with high production of madecassoside

[0132] The following steps were used for cultivation:

[0133] The Centella asiatica stem cell line obtained in Example 1 was placed in a culture medium and stirred for 6 days. The culture conditions were: red-blue light ratio R:B=7:3, light intensity was 100 μmol·m -2 ·s -1 , photoperiod 12h / 12h, stirring speed 100rpm;

[0134] After the cultivation is completed, filtration is performed to obtain the Centella asiatica stem cells with high hydroxymadecassoside production; the content of hydroxymadecassoside in the Centella asiatica stem cells with high hydroxymadecassoside production relative to the dry weight of the Centella asiatica stem cells with high hydroxymadecassoside production is detected, and the results are shown in the results of Example 1 in Table 5.

[0135] The culture medium is based on the B5 liquid culture medium, and 0.05 mmol / L SA (salicylic acid), 20 g / L sucrose, 10 g / L glucose, 0.5 mg / L TDZ and 1.0 mg / L 2,4-D are further added based on the volume of the B5 culture medium. The pH of the culture medium is 6.0 (the pH is adjusted with an aqueous NaOH solution and / or HCl).

[0136] Embodiment 3: the investigation of disinfection time

[0137] The time for oscillating and immersing the stem tip of the Centella asiatica seedlings in a 1wt% NaClO aqueous solution for disinfection was investigated. The specific investigation time is shown in Table 1. The remaining operations are the same as in Example 1. Then, the effects of different disinfection times on the contamination rate and induction rate are calculated. The results are shown in Table 1.

[0138] Table 1: Effects of different disinfection times of 1% NaClO on contamination rate and induction rate

[0139]

[0140]

[0141] Conclusion: The contamination rate of explants soaked and disinfected for only 4 minutes with 1wt% NaClO solution reached 70%, while the explants soaked and disinfected for 8 minutes showed no contamination and had the highest induction rate. The explants soaked and disinfected for 10 minutes and 15 minutes showed no contamination, but the induction rate dropped significantly. When using sodium hypochlorite to disinfect plant explants, appropriate disinfection time is crucial to balance cell protection and complete killing of microorganisms. Too long time may cause cell damage, nutrient absorption disorders, reduced survival rate, increased risk of mutation and prolonged culture cycle, while too short time may lead to incomplete disinfection, increase the risk of contamination, and thus be unfavorable for induction. Therefore, the optimal time for soaking and disinfecting the stem tip explants of Centella asiatica with 1wt% NaClO solution is 8 minutes, which is beneficial to avoid contamination and improve the induction rate, and has unexpected technical effects.

[0142] Example 4: Investigation of the basal medium of the induction medium

[0143] The basal medium in the induction medium was investigated based on the induction medium of Example 1. The B5 medium in the induction medium was replaced with MS medium, N6 medium or WPM medium, respectively. The remaining operations were the same as in Example 1. The induction rates of the induction medium with different basal mediums were detected. The results are shown in Table 2.

[0144] Table 2: Investigation of the basal medium for induction culture

[0145] Basic culture medium Induction rate (%) MS medium 84 N6 medium 62 WPM medium 46

[0146] Conclusion: Compared with other basal culture media, the use of B5 basal culture medium is more conducive to improving the induction rate and has unexpected technical effects.

[0147] Example 5: Investigation of auxin in induction medium

[0148] Based on the induction medium of Example 1, the auxin in the induction medium was examined (the selection of auxin is shown in Table 3, and the concentration of the replaced auxin is the same as the mass concentration of the original auxin). Except for the selection of auxin, the other operations were the same as those of Examples 1 and 2. The induction rates of the induction medium containing different auxins were detected, and the results are shown in Table 3.

[0149] Table 3: Results of the investigation of auxin in the induction medium

[0150]

[0151] Conclusion: Compared with other auxins, the addition of indoleacetic acid to the induction medium has an unexpected technical effect of increasing the induction rate.

[0152] Example 6: Investigation of cytokinins in induction medium

[0153] Based on the induction medium of Example 1, the cytokinins in the induction medium were examined (the selection of cytokinins is shown in Table 4, and the concentration of the replaced cytokinins is the same as the mass concentration of the original cytokinins). Except for the selection of cytokinins, the remaining operations were the same as those of Examples 1 and 2. The induction rates of the induction medium containing different cytokinins were detected, and the results are shown in Table 4.

[0154] Table 4: Results of investigation of cytokinins in induction medium

[0155]

[0156]

[0157] Conclusion: Compared with other cytokinins, the addition of thidiazuron to the induction medium has an unexpected technical effect of increasing the induction rate.

[0158] Example 7: Investigation of auxin in proliferation medium A

[0159] Based on the proliferation medium A of Example 1, the auxin in the proliferation medium A was examined (the selection of auxin is shown in Table 5, and the mass concentration of the replaced auxin is the same as that of the original auxin). Except for the selection of auxin, the remaining operations were the same as those of Examples 1 and 2. The proliferation multiples (defined as the proliferation multiples in Example 1) of the induction medium A containing different auxins and the content of madecassoside in the dry weight of the Centella asiatica stem cells with high madecassoside production obtained after culturing in the culture medium using the culturing method of Example 2 were detected, and the results are shown in Table 5.

[0160] Table 5: Results of the study on auxin in proliferation medium A

[0161]

[0162] Conclusion: Compared with the use of other auxins, the addition of 2,4-dichlorophenoxyacetic acid in proliferation medium A has an unexpected technical effect of increasing the proliferation multiple and increasing the content of madecassoside in the high-yielding Centella asiatica stem cells obtained by subsequent culture in culture medium.

[0163] Example 8: Investigation of cytokinins in proliferation medium A

[0164] Based on the proliferation medium A of Example 1, the cytokinins in the proliferation medium A were investigated (the selection of cytokinins is shown in Table 6, and the concentration of the replaced cytokinins is the same as the mass concentration of the original cytokinins). Except for the selection of cytokinins, the remaining operations were the same as those of Examples 1 and 2. The proliferation multiples (defined as the proliferation multiples in Example 1) of the induction medium A containing different cytokinins and the content of madecassoside in the dry weight of the Centella asiatica stem cells with high madecassoside production obtained after culturing in the culture medium using the culture method of Example 2 were detected, and the results are shown in Table 6.

[0165] Table 6: Results of investigation of cytokinins in proliferation medium A

[0166]

[0167] Conclusion: Compared with other cytokinins, the addition of thidiazuron to proliferation medium A has an unexpected technical effect of increasing the proliferation multiple and the content of madecassoside in the high-yielding Centella asiatica stem cells obtained by subsequent culture in culture medium.

[0168] Example 9-Example 10: Investigation of proliferation culture method

[0169] Based on the method of Example 1, the proliferation medium A in step (3) was replaced by the proliferation medium B (i.e., only the proliferation medium B was used for 10 days of culture and then the new proliferation medium B was used for 10 days of culture, which was recorded as Example 9); and the proliferation medium B in step (3) was replaced by the proliferation medium A (i.e., only the proliferation medium A was used for 10 days of culture and then the new proliferation medium A was used for 10 days of culture, which was recorded as Example 10), and the rest of the operations were the same as those in Example 1 and Example 2. The proliferation multiples (defined as the proliferation multiples in Example 1) of different proliferation culture methods and the content of madecassoside in the dry weight of the high-yielding Centella asiatica stem cells obtained after culture in the culture medium using the culture method of Example 2 were detected, and the results are shown in Table 7.

[0170] Table 7: Investigation of different proliferation culture methods

[0171]

[0172] Conclusion: Compared with the use of a single proliferation culture medium, the present invention adopts alternating culture medium A and proliferation culture medium B, which has an unexpected technical effect of increasing the proliferation multiple and the content of madecassoside in the dry weight of Centella asiatica stem cells with high madecassoside production obtained after culture in the culture medium.

[0173] Example 11: Investigation of inducers in culture medium

[0174] Based on the culture medium of Example 2, the inducer in the culture medium of Example 2 was investigated, and the inducer in the culture medium of Example 2 was replaced with other inducers (see Table 8 for details, the concentration of the replaced inducer was the same as the molar concentration of the original inducer), and except for the difference in the inducer, the remaining operations were the same as those of Example 1 and Example 2. The content of madecassoside in the dry weight of the Centella asiatica stem cells with high madecassoside production obtained after culturing in the culture medium using the culture method of Example 2 was detected, and the results are shown in Table 8.

[0175] Table 8: Investigation of inducers in culture medium

[0176]

[0177] Conclusion: Compared with the use of other inducers, the addition of salicylic acid to the culture medium of the present invention is more conducive to increasing the content of madecassoside in the Centella asiatica stem cells with high madecassoside production obtained after culture in the culture medium, and has unexpected technical effects.

[0178] Example 12: Investigation of the ratio of red and blue light

[0179] Based on the method of Example 2, the effects of different red-blue light ratios on the content of madecassoside in the Centella asiatica stem cells with high madecassoside production obtained after culture in the culture medium were investigated (different red-blue light ratios are shown in Table 9). The remaining operations were the same as those of Examples 1 and 2. The content of madecassoside in the dry weight of the Centella asiatica stem cells with high madecassoside production obtained after culture in the culture medium using the culture method of Example 2 was detected. The results are shown in Table 9.

[0180] Table 9: Investigation of red and blue light ratio

[0181]

[0182]

[0183] Conclusion: Compared with other red-blue light ratios, the red-blue light ratio R:B=7:3 of the present invention is more conducive to increasing the content of madecassoside in the Centella asiatica stem cells with high madecassoside production obtained after culture in the culture medium, and has unexpected technical effects.

[0184] Example 13-Example 15: Investigation of different contents of components in culture medium

[0185] Based on the culture medium of Example 2, the effects of different contents of components in the culture medium were investigated. Except for the different concentrations of the components in the culture medium, the rest of the operations were the same as those in Example 1 and Example 2. The content of hydroxymadecassoside in the dry weight of Centella asiatica stem cells obtained after culture in the culture medium using the culture method of Example 2 was detected. The results are shown in Table 10.

[0186] The culture medium of Example 13 (the glucose in the culture medium of Example 2 is replaced with sucrose): on the basis of the B5 liquid culture medium, 0.05 mmol / L of SA, 30 g / L of sucrose, 0.5 mg / L of TDZ and 1.0 mg / L of 2,4-D are additionally added based on the volume of the B5 liquid culture medium, and the pH of the culture medium is 6.0 (the pH is adjusted with NaOH aqueous solution and / or HCl).

[0187] The culture medium of Example 14 (the glucose concentration in the culture medium of Example 2 was changed to 5 g / L, and the sucrose concentration was changed to 25 g / L): on the basis of the B5 liquid culture medium, 0.05 mmol / L of SA, 25 g / L of sucrose, 5 g / L of glucose, 0.5 mg / L of TDZ and 1.0 mg / L of 2,4-D were additionally added, based on the volume of the B5 liquid culture medium, and the pH of the culture medium was 6.0 (the pH was adjusted with NaOH aqueous solution and / or HCl).

[0188] The culture medium of Example 15 (without adding SA): On the basis of the B5 liquid culture medium, 20 g / L of sucrose, 10 g / L of glucose, 0.5 mg / L of TDZ and 1.0 mg / L of 2,4-D were further added, based on the volume of the B5 liquid culture medium, and the pH of the culture medium was 6.0 (the pH was adjusted with NaOH aqueous solution and / or HCl).

[0189] Table 10: Investigation of different contents of components in culture medium

[0190]

[0191] Conclusion: Compared with the culture medium without SA addition, the culture medium with SA added in the present invention can greatly increase the content of madecassoside in the obtained Centella asiatica stem cells with high madecassoside production, which has unexpected technical effects.

[0192] Example 16: Centella asiatica stem cell freeze-dried powder

[0193] The Centella asiatica stem cells with high production of madecassoside obtained in Example 2 were pre-frozen at -80°C for 4 hours and then vacuum freeze-dried at -55°C for 24 hours to obtain the Centella asiatica stem cell freeze-dried powder.

[0194] Test Example 1:

[0195] The lyophilized powder prepared from the Centella asiatica stem cells of the present invention can be used as a raw material with anti-inflammatory, soothing and other skin care effects. The Centella asiatica stem cell lyophilized powder obtained in Example 16 was subjected to soothing-LPS-stimulated macrophage-inflammatory factor detection (TNF-α, IL-1β).

[0196] Procedure: This experiment is based on an in vitro evaluation model for soothing efficacy. The soothing efficacy is evaluated by using an LPS-stimulated macrophage model. The soothing efficacy of the sample is evaluated by detecting changes in the content of related inflammatory factors after the sample acts on macrophages. The content of inflammatory factors TNFα and IL-1β is detected according to the operating instructions of the Mouse TNF-α ELISA kit and the operating instructions of the Mouse IL-1β ELISA kit, respectively.

[0197] Table 11: Experimental design

[0198]

[0199]

[0200] Results: See Table 12 and Table 13.

[0201] Table 12: TNF-α data summary

[0202]

[0203]

[0204] Table 13: IL-1β data summary

[0205] Group Average concentration (pg / mL) SD p-value Blank control (BC) 74.940 3.315 / Negative control (NC) 139.161 1.865 <![CDATA[0.001 ## ]]> Positive Control (PC) 75.732 3.654 <![CDATA[0.001 ** ]]> CA - 0.5% 77.046 3.537 <![CDATA[0.001 ** ]]> CA - 0.1% 93.457 6.989 <![CDATA[0.008 ** ]]>

[0206] Note: When using the t-test two-tailed test method for statistical analysis, the significance of the NC group compared with the BC group is indicated by #, and p-value < 0.05 is indicated by # , p-value<0.01 is indicated as ## The significance of the sample group, PC group and NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0207] in conclusion:

[0208] Compared with the BC group, the secretion of TNF-α, a macrophage inflammatory factor, in the NC group increased significantly (p<0.01), indicating that the LPS stimulation model was successfully established in this experiment. Compared with the NC group, the secretion of TNF-α, a macrophage inflammatory factor, in the PC group was significantly reduced at a concentration of 100 μg / mL of dexamethasone (p<0.01), indicating that the positive control of this experiment was effective. Compared with the NC group, the secretion of TNF-α, a macrophage inflammatory factor, was significantly reduced at a concentration of 0.5% (m / V) and 0.1% (m / V) in the sample (p<0.01), indicating that the sample has anti-inflammatory and soothing effects.

[0209] Compared with the BC group, the secretion of IL-1β, an inflammatory factor of macrophages, in the NC group increased significantly (p<0.01), indicating that the LPS stimulation model was successfully established in this experiment. Compared with the NC group, the secretion of IL-1β, an inflammatory factor of macrophages, in the PC group was significantly reduced at a concentration of 100 μg / mL of dexamethasone (p<0.01), indicating that the positive control of this experiment was effective. Compared with the NC group, the secretion of IL-1β, an inflammatory factor of macrophages, was significantly reduced when the sample was administered at a concentration of 0.5% (m / V) and 0.1% (m / V) (p<0.01). Based on the LPS-stimulated macrophage anti-inflammatory model, the secretion of TNF-α and IL-1β, inflammatory factors of macrophages, was significantly reduced when the sample was administered at a concentration of 0.1% (m / V) and 0.5% (m / V), which was statistically different from the NC group (p<0.01), indicating that the sample has anti-inflammatory and soothing effects.

[0210] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for isolating and culturing Centella asiatica stem cell lines, characterized in that: The following steps are involved: (1) Selection and disinfection of explants: Take the stem tip of a Centella asiatica seedling, disinfect it, and obtain an explant; (2) Isolation and induction of meristem: The stem tip containing meristem in the explant sterilized in step (1) is placed in an induction medium for induction culture to obtain induced Centella asiatica stem cells; the induction medium comprises B5 medium, based on the volume of the B5 medium, 30 g / L sucrose, 6 g / L agar, 1.5 mg / L to 2.0 mg / L indoleacetic acid and 0.5 mg / L to 1 mg / L thidiazuron, and the pH of the induction medium is 5.8-6.2; The induction culture temperature of step (2) is 25°C ± 2°C; The induced culture in step (2) is carried out under dark conditions. The induction culture in step (2) is cultured for 7 to 14 days; (3) proliferation culture: transferring the induced Centella asiatica stem cells obtained in step (2) to a proliferation medium for culture to obtain the Centella asiatica stem cell line; the proliferation medium includes a proliferation medium A and a proliferation medium B; The proliferation medium A comprises, based on the volume of the B5 medium, 20 g / L of sucrose, 10 g / L of glucose, 6 g / L of agar, 0.5 mg / L-1.0 mg / L of thidiazuron, and 1.0 mg / L-2.0 mg / L of 2,4-dichlorophenoxyacetic acid, and the pH of the proliferation medium A is 5.8-6.2; The proliferation medium B comprises, based on the volume of the B5 medium, 20 g / L of sucrose, 10 g / L of glucose, 6 g / L of agar, 0.5 mg / L-1.0 mg / L of naphthylacetic acid, 0.5 mg / L-1.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L-1 mg / L of thidiazuron and 0.5-1 mg / L of 6-benzylaminoadenine, and the pH of the proliferation medium B is 5.8-6.2; The culturing in step (3) includes first culturing with proliferation medium A and then culturing with proliferation medium B; The culture temperature of the proliferation medium A is 25°C ± 2°C; The culture illumination condition for culturing using proliferation medium A is in dark conditions; The culture time of the culture using proliferation medium A is 7 days to 14 days; The culture temperature of the proliferation medium B is 25°C ± 2°C; The culture illumination condition for culturing using proliferation medium B is in dark conditions; The culture time for culturing using proliferation medium B is 7 days to 14 days.

2. The method according to claim 1, wherein the disinfection in step (1) comprises immersing in a 1wt% NaClO aqueous solution by shaking for 4 min to 15 min, rinsing with sterile water for 3 to 5 times, immersing in 75% alcohol by shaking for 30 s, rinsing with sterile water for 3 to 5 times, and absorbing the sterile water with sterile filter paper.

3. The method according to claim 1, wherein the disinfection in step (1) comprises immersing in a 1wt% NaClO aqueous solution by shaking for 8 min, rinsing with sterile water for 3-5 times, immersing in 75% alcohol by shaking for 30 s, rinsing with sterile water for 3-5 times, and absorbing the sterile water with sterile filter paper.

4. The method according to any one of claims 2 to 3, wherein the disinfection further comprises flushing with water for 30 min to 40 min before using the 1 wt % NaClO aqueous solution.

5. A method for culturing Centella asiatica stem cells with high production of madecassoside, characterized in that: include: The Centella asiatica stem cell line obtained by the method according to any one of claims 1 to 4 is cultured in a culture medium, and filtered after the culture is completed to obtain the Centella asiatica stem cell with high madecassoside production.

6. The culture method according to claim 5, wherein the culture solution is based on the B5 liquid culture medium, and 0-0.1 mmol / L of inducer, 20 g / L-30 g / L of sucrose, 0-10 g / L of glucose, 0.5-2.0 mg / L of thidiazuron and 1.0-2.0 mg / L of 2,4-dichlorophenoxyacetic acid are additionally added based on the volume of the B5 liquid culture medium, and the pH of the culture solution is 5.8-6.2; the inducer is salicylic acid, methyl jasmonate, gibberellin, abscisic acid or ethylene.

7. The culture method according to claim 6, wherein the inducer is salicylic acid.

8. The culture method according to claim 5, wherein the illumination condition of the culture is a light intensity of 100 μmol·m -2 ·s -1 -200 μmol·m -2 ·s -1 .

9. The culture method according to claim 5, wherein the photoperiod of the culture is 0 h / 24 h-24 h / 0 h.

10. The culture method according to claim 5, wherein the photoperiod of the culture is 12 h / 12 ​​h. The culture method according to claim 5 , wherein the culture time is 4 to 6 days.

12. The culture method according to claim 5, wherein the stirring speed of the culture is 50 rpm-150 rpm. The culture method according to claim 5 , wherein the stirring speed of the culture is 100 rpm.

14. The culture method according to claim 5, wherein the ratio of red light to blue light in the culture illumination is R:B=7:

3.

15. An induction culture medium for inducing Centella asiatica meristems to obtain Centella asiatica stem cells, characterized in that: The induction medium is based on the B5 medium, and 30 g / L of sucrose, 6 g / L of agar, 1.5 mg / L-2.0 mg / L of indoleacetic acid and 0.5 mg / L-1 mg / L of thidiazuron are further added based on the volume of the B5 medium. The pH of the induction medium is 5.8-6.

2.

16. A Centella asiatica stem cell freeze-dried powder, characterized in that: The preparation method of the Centella asiatica stem cell freeze-dried powder comprises: freeze-drying the Centella asiatica stem cells with high production of madecassoside obtained by the culturing method according to any one of claims 5 to 14 to obtain the Centella asiatica stem cell freeze-dried powder.

17. A Centella asiatica stem cell, which is selected from the Centella asiatica stem cell line obtained by culturing according to any one of claims 1 to 4 or the Centella asiatica stem cell with high madecassoside production obtained by culturing according to any one of claims 5 to 14.

18. Use of the Centella asiatica stem cells with high production of madecassoside obtained by the culture method according to any one of claims 5 to 14 or the Centella asiatica stem cell freeze-dried powder according to claim 16 in the preparation of cosmetics with anti-inflammatory or soothing effects.

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