Preparation method and application of centella asiatica stem cells for high-yield madecassoside

Abstract

The invention provides a preparation method and application of centella asiatica stem cells for high-yield madecassoside, and belongs to the technical field of stem cells. The preparation method comprises the steps of disinfection, induction, proliferation, culture of centella asiatica stem cells with high madecassoside yield and the like, and has the unexpected technical effects that the pollution rate is low, the induction rate is high, the proliferation multiple is high, and the madecassoside content in the obtained centella asiatica stem cells with high madecassoside yield is high. The product is beneficial to industrial production and can be used as a skin care component in a product with anti-inflammatory, soothing and other effects.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and particularly relates to a method for preparing Centella asiatica stem cells with high yield of madecassoside and its use. Background Art

[0002] Centella asiatica (L.) Urban. is a perennial creeping herb of the genus Centella in the family Apiaceae, growing in humid areas, such as rural roadside, beside small rivers and ditches, etc., and is widely distributed in tropical and subtropical regions of the world. In China, its distribution range is mainly concentrated in the areas south of the Yangtze River. Centella asiatica is a commonly used traditional 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 promoting diuresis, and detumescence and detoxification. It is applicable to symptoms such as damp-heat, diarrhea, stranguria with blood, and sore toxin. Clinically, it is also applied to the treatment of diseases such as traumatic injury and skin diseases. Its mechanism of action includes promoting the proliferation of fibroblasts, increasing the synthesis of collagen and the content of fibronectin in cells, improving the tensile strength of new skin, and inhibiting the inflammatory phase of hypertrophic scars and keloids.

[0003] Experimental research results show that the active compounds of Centella asiatica include pentacyclic triterpenoids, such as madecassoside, asiaticoside, madecassic acid, asiatic acid, etc. Among them, madecassoside is the main active ingredient in Centella asiatica, and has the effects of promoting wound healing, repairing skin scars, anti-allergy, anti-inflammation, whitening, antioxidant, anti-gastric ulcer and treating hepatitis, etc. Centella asiatica has been widely used in the fields of medicine, health food and beauty, etc., and has a broad market prospect. However, 90% of the global Centella asiatica raw materials rely on wild resources. Due to the urbanization process and the use of herbicides, Centella asiatica is rarely seen around the living environment and in the surrounding farmland and mountains. Therefore, introduction and cultivation, artificial breeding, etc. have become new ways to effectively utilize Centella asiatica resources.

[0004] However, it is difficult to industrially develop physiologically active substances, and the main reasons are as follows: 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 specific organs of plants in small amounts; it involves environmental problems related to natural destruction; the chemical structure of physiologically active substances derived from plants is very complex and requires multiple polymerization processes, resulting in 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 comprehensively evaluated the quality of 29 batches of Centella asiatica from different origins and found that the content of madecassoside in wild Centella asiatica was 0.541% - 1.915% (see "Liu Fusong, Li Yaoyao, Nie Xuqiang, et al. Evaluation of the Quality of Centella asiatica Based on Sensory Characteristics Combined with Grey Relational Analysis Method [J]. Chinese Journal of New Drugs, 2021, 30(20): 1881 - 1889."). Therefore, the content of madecassoside in wild Centella asiatica is low and it is difficult to be industrially applied.

[0006] Plants are an important source for the production of a large number of natural products. A common strategy for obtaining natural products is to extract them from raw plants. However, the resources and growth rate of plants usually limit the yield of natural products. As one of the bioengineering technologies, plant cell culture methods have long been evaluated as the most ideal technology for providing useful substances derived from plants without causing environmental problems. Plant stem cell culture technology has been introduced for the large-scale production of many useful components. A variety of plant-derived compounds are used in various fields such as pharmaceuticals, foods, and cosmetics. Producing useful substances through plant stem cell culture technology has many advantages compared to directly extracting useful substances from plants. In particular, different from the 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, thus solving prominent problems such as ecosystem damage. Moreover, plant stem cells also have characteristics such as a short growth cycle, a 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, a low stem cell proliferation multiple, and a low content of madecassoside in the obtained Centella asiatica stem cells.

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

[0009] To solve the above technical problems, the present invention provides the following technical solutions.

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

[0011] A method for separating and culturing a Centella asiatica stem cell line, which comprises the following steps:

[0012] (1) Selection and disinfection of explants: Take the stem tip of Centella asiatica seedlings, disinfect, and obtain explants;

[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 is based on the B5 medium. 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 NAA (naphthaleneacetic acid), 0.5 mg / L - 1.0 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L - 1 mg / L of TDZ, and 0.5 - 1 mg / L of 6-BA (6-benzylaminopurine) are additionally added. 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. 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 of TDZ (thidiazuron), and 1.0 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid) are additionally added. The pH of the proliferation medium A is 6.0.

[0022] In some embodiments, the proliferation medium B is based on the B5 medium. 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 of NAA (naphthaleneacetic acid), 0.5 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L of TDZ, and 0.5 mg / L of 6-BA (6-benzylaminopurine) are additionally added. The pH of the proliferation medium B is 6.0.

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

[0024] In some embodiments, the pH of the proliferation medium B is adjusted using an aqueous NaOH solution and / or HCl, 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 culture in step (3) includes first culturing with the proliferation medium A and then changing to the proliferation medium B for culturing.

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

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

[0030] In some embodiments, the culture time for culturing with proliferation medium A is 7 days to 14 days. In some embodiments, the culture time for culturing with 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 with proliferation medium B is 25°C ± 2°C.

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

[0033] In some embodiments, the culture time for culturing with proliferation medium B is 7 days to 14 days. In some embodiments, the culture time for culturing with 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 for the induction culture in step (2) is 25°C ± 2°C.

[0035] In some embodiments, the culture light condition for the induction culture in step (2) is under dark conditions.

[0036] In some embodiments, the culture time for the induction culture in step (2) is 7 days to 14 days. In some embodiments, the culture time for the induction 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 in step (1) includes shaking and soaking in 1 wt% NaClO aqueous solution for 4 min - 15 min (such as 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min), rinsing with sterile water 3 - 5 times, shaking and soaking in 75% alcohol for 30 s, rinsing with sterile water 3 - 5 times, and blotting dry with sterile filter paper.

[0038] In some preferred embodiments, the disinfection in step (1) includes shaking and soaking in 1 wt% NaClO aqueous solution for 8 min, rinsing with sterile water 3 - 5 times, shaking and soaking in 75% alcohol for 30 s, rinsing with sterile water 3 - 5 times, and blotting dry with sterile filter paper.

[0039] In some embodiments, the disinfection further includes rinsing with water for 30 min - 40 min before using 1 wt% aqueous NaClO 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 production of madecassoside, which includes: placing the Centella asiatica stem cell line obtained by the method of the first aspect in a culture medium for culturing, and filtering after the culturing is completed to obtain the Centella asiatica stem cells with high production of madecassoside.

[0042] In some preferred embodiments, the culture medium is based on B5 liquid medium. Based on the volume of B5 liquid medium, additionally add 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. 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 B5 liquid medium. Based on the volume of B5 liquid medium, additionally add 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. The pH of the culture medium is 6.0.

[0044] In some embodiments, the culture medium is based on B5 liquid medium. Based on the volume of B5 liquid medium, additionally add 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. The pH of the culture medium is 6.0.

[0045] In some preferred embodiments, the culture medium is based on B5 liquid medium, and additionally add 0.05 mmol / L of SA (salicylic acid), 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. The pH of the culture medium is 6.0.

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

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

[0048] In some embodiments, the light condition for the cultivation is that the light intensity is 100 μmol·m -2 ·s -1 -200 μmol·m -2 ·s -1 。In some embodiments, the light condition for the cultivation is that the light intensity is 100 μmol·m -2 ·s -1 、110 μmol·m -2 ·s -1 、120 μmol·m -2 ·s -1 、130 μmol·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 light cycle for the cultivation is 0 h / 24 h - 24 h / 0 h. In some embodiments, the light cycle for the cultivation is 12 h / 12 h (indicating that the light-dark alternation pattern is 12 h of light and 12 h of darkness). In some embodiments, the light cycle for the cultivation is 0 h / 24 h (indicating that the light-dark alternation pattern is 0 h of light and 24 h of darkness), 6 h / 18 h (indicating that the light-dark alternation pattern is 6 h of light and 18 h of darkness), 12 h / 12 h (indicating that the light-dark alternation pattern is 12 h of light and 12 h of darkness), 18 h / 6 h (indicating that the light-dark alternation pattern is 18 h of light and 6 h of darkness), or 24 h / 0 h (indicating that the light-dark alternation pattern is 24 h of light and 0 h of darkness). In some embodiments, the light cycle for the cultivation is 12 h / 12 h (indicating that the light-dark alternation pattern is 12 h of light and 12 h of darkness).

[0050] In some embodiments, the cultivation time for the cultivation is 4 days - 6 days. In some embodiments, the cultivation time for the cultivation is 4 days, 5 days, or 6 days.

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

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

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

[0054] An induction medium for inducing the meristem of Centella asiatica to obtain Centella asiatica stem cells, the induction medium is based on the B5 medium, and based on the volume of the B5 medium, additionally add 30 g / L of sucrose, 6 g / L of agar, 1.5 mg / L - 2.0 mg / L of IAA (indole-3-acetic acid) and 0.5 mg / L - 1 mg / L of TDZ (thidiazuron), and the pH of the induction medium is 5.8 - 6.2.

[0055] In some embodiments, the induction medium includes, based on the B5 medium, based on the volume of the B5 medium, add 30 g / L of sucrose, 6 g / L of agar, 1.5 mg / L of IAA (indole-3-acetic acid) and 0.5 mg / L of TDZ (thidiazuron), 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 with an aqueous sodium hydroxide solution and / or hydrochloric acid or with an aqueous NaOH solution and / or an aqueous HCl solution.

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

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

[0060] The proliferation medium A is based on the B5 medium. 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) are additionally added. The pH of the proliferation medium A is 5.8 - 6.2;

[0061] The proliferation medium B is based on the B5 medium. 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 NAA (naphthaleneacetic acid), 0.5 mg / L - 1 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L - 1 mg / L of TDZ, and 0.5 - 1 mg / L of 6-BA (6-benzylaminopurine) are additionally added. 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. 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 of TDZ (thidiazuron), and 1.0 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid) are additionally added. The pH of the proliferation medium A is 6.0.

[0063] In some embodiments, the proliferation medium B is based on the B5 medium. 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 of NAA (naphthaleneacetic acid), 0.5 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L of TDZ, and 0.5 mg / L of 6-BA (6-benzylaminopurine) are additionally added. The pH of the proliferation medium B is 6.0.

[0064] In some embodiments, the pH of the proliferation medium A is adjusted using an aqueous NaOH solution and / or HCl, or using an aqueous NaOH solution and / or an 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 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 solution for culturing centella asiatica stem cells with a high content of madecassoside.

[0069] A culture solution for culturing centella asiatica stem cells with a high content of madecassoside, the culture solution is based on B5 liquid medium, and based on the volume of B5 liquid medium, additionally adding 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, the pH of the culture solution 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 by NaOH aqueous solution and / or HCl or by NaOH aqueous solution and / or HCl aqueous solution.

[0072] In some embodiments, the culture solution is based on B5 liquid medium, and based on the volume of B5 liquid medium, additionally adding 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, the pH of the culture solution is 6.0.

[0073] In some embodiments, the culture solution is based on B5 liquid medium, and based on the volume of B5 liquid medium, additionally adding 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, the pH of the culture solution is 6.0.

[0074] In some preferred embodiments, the culture solution is based on B5 liquid medium, and additionally adding 0.05 mmol / L of SA (salicylic acid), 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, the pH of the culture solution is 6.0.

[0075] In a sixth aspect, the present invention provides a freeze-dried powder of centella asiatica stem cells.

[0076] A centella asiatica stem cell freeze-dried powder, and its preparation method includes: subjecting the centella asiatica stem cells that produce high-yield asiaticoside obtained by the cultivation method described in the second aspect to freeze-drying to obtain the centella asiatica stem cell freeze-dried powder.

[0077] In the 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 the cultivation method described in the first aspect or the centella asiatica stem cells that produce high-yield asiaticoside obtained by the cultivation method described in the second aspect.

[0079] In the eighth aspect, the present invention provides an application of the centella asiatica stem cells that produce high-yield asiaticoside obtained by the cultivation method described in the second aspect or the centella asiatica stem cell freeze-dried powder described in the sixth aspect.

[0080] An application of the centella asiatica stem cells that produce high-yield asiaticoside obtained by the cultivation method described in the second aspect or the centella asiatica stem cell freeze-dried powder described in the sixth aspect in the preparation of cosmetics for anti-inflammatory or soothing purposes.

[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 soaking and disinfecting the centella asiatica stem tip explant with 1 wt% NaClO aqueous solution in the present invention is 8 minutes, which is beneficial for avoiding contamination and can also improve the induction rate, having an unexpected technical effect.

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

[0085] (3) Compared with using other cytokinins, adding thidiazuron to the induction medium has an unexpected technical effect of improving the induction rate.

[0086] (4) Compared with using other auxins, adding 2,4-dichlorophenoxyacetic acid to the proliferation medium A has unexpected technical effects of increasing the proliferation multiple and increasing the content of asiaticoside in the centella asiatica stem cells that produce high-yield asiaticoside obtained by subsequent cultivation in the culture solution.

[0087] (5) Compared with using other cytokinins, adding thidiazuron to the proliferation medium A has unexpected technical effects of increasing the proliferation multiple and increasing the content of asiaticoside in the centella asiatica stem cells that produce high-yield asiaticoside obtained by subsequent cultivation in the culture solution.

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

[0089] (7) Compared with using other inducers, adding salicylic acid to the culture solution of the present invention is more conducive to increasing the content of asiaticoside in the centella asiatica stem cells with high asiaticoside production obtained after culturing with the culture solution, and has an unexpected technical effect.

[0090] (8) Compared with using other red-blue light ratios, using the red-blue light ratio of R:B = 7:3 of the present invention is more conducive to increasing the content of asiaticoside in the centella asiatica stem cells with high asiaticoside production obtained after culturing with the culture solution, and has an unexpected technical effect.

[0091] (9) Compared with other basal media, using B5 basal medium is more conducive to increasing the induction rate, and has an unexpected technical effect.

[0092] (10) Compared with using a culture solution without SA, the present invention uses a culture solution added with SA, which can greatly increase the content of asiaticoside in the stem cells after culture, and has an unexpected technical effect.

[0093] (11) The content of asiaticoside in the centella asiatica stem cells with high asiaticoside production prepared by the present invention is high. The obtained centella asiatica stem cells with high asiaticoside production have anti-inflammatory, soothing and other effects, and can be used as components in products with anti-inflammatory, soothing and other effects.

[0094] Term Explanation

[0095] In the foregoing of the present invention, whether or not words such as "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the numerical value of each number may have a difference of less than ±10% or a reasonable difference considered by those skilled in the art, such as a difference of ±1%, ±2%, ±3%, ±4% or ±5%.

[0096] The term "optionally", "optional" or "optionally" means that the subsequent described event or situation may but does not necessarily occur.

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

[0098] The term "wt%" represents mass percentage.

[0099] Induction rate (%) = (number of explants generating 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[NH 4 SO 4 > 134 <![CDATA[MnSO 4 ·H 2 O]]> 10 CaCl2 113.23 <![CDATA[Na2MoO 4 ·2H 2 O]]> 0.25 <![CDATA[MgSO 4 > 121.56 KI 0.75 <![CDATA[KNO 3 > 2500 <![CDATA[ZnSO 4 ·7H 2 O]]> 2 <![CDATA[Sodium hydride 2 Phosphorus monoxide 4 > 130.44 moy-Inositol 100 <![CDATA[H 3 BO 3 > 3 Nicotinic acid 1 <![CDATA[CoCl 2 ·6H 2 O]]> 0.025 Pyridoxine HCl 1 <![CDATA[CuSO 4 ·5H 2 O]]> 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” 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 mode: isocratic elution;

[0116] e) Injection volume: 10 μL;

[0117] f) Column temperature: 25 °C;

[0118] g) Wavelength: 205 nm.

[0119] Madecassoside reference solution: Accurately weigh 10.0 mg of madecassoside reference substance in a 10 mL volumetric flask, and then make up the volume to the mark with methanol. That is, a standard stock solution with a mass concentration of 1.0 mg / mL is obtained and stored at -20 °C.

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

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

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

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

[0124] (1) Selection and disinfection of explants: Take the stem tip of Centella asiatica seedlings, disinfect to obtain explants; the disinfection includes: first rinse with water for 30 min; then soak and oscillate in 1 wt% NaClO aqueous solution for 8 min, rinse 3 times with sterile water, soak and oscillate in 75% alcohol for 30 s, rinse 3 times with sterile water, and use sterile filter paper to absorb the sterile water;

[0125] (2) Isolation and induction of meristem-containing: Peel the meristem-containing stem tip from the explants disinfected in step (1), and place the meristem-containing stem tip in the induction medium and conduct induction culture at 25 °C under dark conditions 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, filter to obtain the Centella asiatica stem cells with high production of madecassoside; detect the content of madecassoside in the Centella asiatica stem cells with high production of madecassoside accounting for the dry weight of the Centella asiatica stem cells with high production of madecassoside, and the results are shown in Example 1 of Table 5.

[0135] The culture medium is based on the B5 liquid medium. Based on the volume of the B5 medium, additionally add 0.05 mmol / L of SA (salicylic acid), 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. The pH of the culture medium is 6.0 (adjust the pH with NaOH aqueous solution and / or HCl).

[0136] Example 3: Investigation of disinfection time

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

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

[0139] Treatment Treatment time (min) Pollution rate (%) Induction rate (%) 1 4 70 10 2 6 5 90 3 8 0 98 4 10 0 72 5 15 0 16

[0140] Conclusion: The contamination rate of the explants soaked and disinfected in 1 wt% NaClO aqueous solution for only 4 min reaches 70%. The explants soaked and disinfected for 8 min show no contamination and have the highest induction rate. The explants soaked and disinfected for 10 min and 15 min have no contamination, but the induction rate drops significantly. When using sodium hypochlorite for plant explant disinfection, an appropriate disinfection time is crucial for balancing cell protection and thorough killing of microorganisms. Too long a time may cause cell damage, nutrient absorption disorders, reduced survival rate, increased mutation risk, and extended culture period, while too short a time may lead to incomplete disinfection and increased contamination risk, thus being unfavorable for induction. Therefore, the optimal time for soaking and disinfecting the stem tip explants of Centella asiatica in 1 wt% NaClO aqueous solution is 8 min, which is beneficial for avoiding contamination and improving the induction rate, and has unexpected technical effects.

[0141] Example 4: Investigation of the basal medium of the induction medium

[0142] Based on the induction medium of Example 1, investigate the basal medium in the induction medium. Replace the B5 medium in the induction medium with MS medium, N6 medium, or WPM medium respectively, and the remaining operations are the same as in Example 1. Detect the induction rates of the induction media with different basal media, and the results are shown in Table 2.

[0143] Table 2: Investigation of the basal medium of the induction medium

[0144]

[0145]

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

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

[0148] Based on the induction medium of Example 1, the auxin in the induction medium was investigated (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 different selection of auxin, the remaining operations were the same as those in Example 1 and Example 2. The induction rates of the induction media containing different auxins were detected, and the results are shown in Table 3.

[0149] Table 3: Investigation results of auxin in the induction medium

[0150] Auxin in the induction medium Induction rate (%) Example 1 100 Replace the indole-3-acetic acid in the induction medium of Example 1 with IBA (3-Indolebutyric acid) 92 Replace the indole-3-acetic acid in the induction medium of Example 1 with NAA (Naphthaleneacetic acid) 82 Replace the indole-3-acetic acid in the induction medium of Example 1 with 2,4-D (2,4-Dichlorophenoxyacetic acid) 88

[0151] Conclusion: Compared with using other auxins, adding indoleacetic acid to the induction medium has unexpected technical effects in improving the induction rate.

[0152] Example 6: Investigation of cytokinin in the induction medium

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

[0154] Table 4: Investigation results of cytokinin in the induction medium

[0155] Cytokinin in the induction medium Induction rate (%) Example 1 100 Replace the thidiazuron in the induction medium of Example 1 with KT (Kinetin) 86 Replace the thidiazuron in the induction medium of Example 1 with 6-BA (6-Benzylaminopurine) 74

[0156] Conclusion: Compared with using other cytokinins, adding thidiazuron to the induction medium has unexpected technical effects in improving the induction rate.

[0157] Example 7: Investigation of auxin in proliferation medium A

[0158] Based on the proliferation medium A of Example 1, the auxins in the proliferation medium A were investigated (the selection of auxins 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 different selection of auxins, the remaining operations were the same as those in Example 1 and Example 2. The proliferation multiples of the induction medium A containing different auxins (defined in the same way as the proliferation multiple in Example 1) and the content of madecassoside in the dry weight of Centella asiatica stem cells with high-yield madecassoside obtained after culturing in the culture solution using the culture method of Example 2 were detected, and the results are shown in Table 5.

[0159] Table 5: Investigation results of auxins in proliferation medium A

[0160]

[0161]

[0162] Conclusion: Compared with the use of other auxins, adding 2,4-dichlorophenoxyacetic acid to the proliferation medium A has the unexpected technical effects of improving the proliferation multiple and increasing the content of madecassoside in the Centella asiatica stem cells with high-yield madecassoside obtained after subsequent culturing in the culture solution.

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

[0165] Table 6: Investigation results of cytokinins in proliferation medium A

[0166]

[0167]

[0168] Conclusion: Compared with the use of other cytokinins, adding thidiazuron to the proliferation medium A has the unexpected technical effects of improving the proliferation multiple and increasing the content of madecassoside in the Centella asiatica stem cells with high-yield madecassoside obtained after subsequent culturing in the culture solution.

[0169] Examples 9 - 10: Investigation of proliferation culture methods

[0170] Based on the method of Example 1, respectively examine replacing the proliferation medium A in step (3) with proliferation medium B (that is, culturing only with proliferation medium B for 10 days and then changing to a new proliferation medium B and continuing to culture for another 10 days, denoted as Example 9); and replacing the proliferation medium B in step (3) with proliferation medium A (that is, culturing only with proliferation medium A for 10 days and then changing to a new proliferation medium A and continuing to culture for another 10 days, denoted as Example 10), and the remaining operations are the same as those in Example 1 and Example 2. Detect the proliferation multiples of different proliferation culture methods (defined in the same way as the proliferation multiple in Example 1) and the content of madecassoside in the dry weight of Centella asiatica stem cells with high-yield madecassoside obtained after culturing with the culture medium in the culture method of Example 2. The results are shown in Table 7.

[0171] Table 7: Investigation of different proliferation culture methods

[0172]

[0173] Conclusion: Compared with using a single proliferation medium, the present invention adopts alternating culture with proliferation medium A and proliferation medium B, which has the unexpected technical effect of improving the proliferation multiple and the content of madecassoside in the dry weight of Centella asiatica stem cells with high-yield madecassoside obtained after culturing with the culture medium.

[0174] Example 11: Investigation of the inducer in the culture medium

[0175] Based on the culture medium of Example 2, investigate the inducer. Respectively replace the inducer in the culture medium of Example 2 with other inducers (specifically shown in Table 8, and the concentration of the replaced inducer is the same as the molar concentration of the original inducer). Except for the difference in the inducer, the remaining operations are the same as those in Example 1 and Example 2. Detect the content of madecassoside in the dry weight of Centella asiatica stem cells with high-yield madecassoside obtained after culturing with the culture medium in the culture method of Example 2. The results are shown in Table 8.

[0176] Table 8: Investigation of the inducer in the culture medium

[0177]

[0178] Conclusion: Compared with using other inducers, adding salicylic acid to the culture medium of the present invention is more conducive to increasing the content of madecassoside in Centella asiatica stem cells with high-yield madecassoside obtained after culturing with the culture medium, and has an unexpected technical effect.

[0179] Example 12: Investigation of the red and blue light ratio

[0180] Based on the method of Example 2, investigate the effect of different red-blue light ratios on the content of asiaticoside in centella asiatica stem cells with high asiaticoside production obtained after culturing in the culture medium (different red-blue light ratios are shown in Table 9). The remaining operations are the same as those in Example 1 and Example 2. The detection measures the content of asiaticoside in the centella asiatica stem cells with high asiaticoside production obtained after culturing in the culture medium, accounting for the dry weight of the stem cells, and the results are shown in Table 9.

[0181] Table 9: Investigation of red-blue light ratios

[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 asiaticoside in centella asiatica stem cells with high asiaticoside production obtained after culturing in the culture medium, and has unexpected technical effects.

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

[0185] Based on the culture medium of Example 2, investigate the effects of different contents of components in the culture medium. Except for the different component concentrations of the culture medium, the remaining operations are the same as those in Example 1 and Example 2. The detection measures the content of asiaticoside in the centella asiatica stem cells with high asiaticoside production obtained after culturing in the culture medium, accounting for the dry weight of the stem cells, and the results are shown in Table 10.

[0186] The culture medium of Example 13 (replacing glucose in the culture medium of Example 2 with sucrose): On the basis of the B5 liquid medium, calculated by the volume of the B5 liquid medium, additionally add 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. The pH of the culture medium is 6.0 (adjusted with NaOH aqueous solution and / or HCl).

[0187] The culture medium of Example 14 (changing the glucose concentration in the culture medium of Example 2 to 5 g / L and the sucrose concentration to 25 g / L): On the basis of the B5 liquid medium, calculated by the volume of the B5 liquid medium, additionally add 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. The pH of the culture medium is 6.0 (adjusted with NaOH aqueous solution and / or HCl).

[0188] Culture medium of Example 15 (without SA addition): Based on the volume of the B5 liquid 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 additionally added. The pH of the culture medium was 6.0 (adjusted with NaOH aqueous solution and / or HCl).

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

[0190]

[0191] Conclusion: Compared with the culture medium without SA addition, the culture medium with SA addition in the present invention can greatly increase the content of asiaticoside in the centella asiatica stem cells that produce high-yield asiaticoside, showing unexpected technical effects.

[0192] Example 16: Freeze-dried powder of centella asiatica stem cells

[0193] The centella asiatica stem cells that produce high-yield asiaticoside obtained in Example 2 were pre-frozen at -80 °C for 4 h and then vacuum freeze-dried at -55 °C for 24 h to obtain the freeze-dried powder of centella asiatica stem cells.

[0194] Test Example 1:

[0195] The freeze-dried powder obtained by preparing the centella asiatica stem cells of the present invention can be used as a raw material with skin care effects such as anti-inflammatory and soothing. The freeze-dried powder of centella asiatica stem cells obtained in Example 16 was subjected to detection of soothing - LPS-stimulated macrophages - inflammatory factors (TNF-α, IL-1β).

[0196] Operation: Based on the in vitro evaluation model of soothing efficacy, the LPS-stimulated macrophage model was used to evaluate the soothing efficacy. By detecting the changes in the content of relevant inflammatory factors after the sample acted on macrophages, the soothing efficacy of the test sample was evaluated. The detection of the contents of inflammatory factors TNFα and IL-1β was carried out according to the operation manuals of the Mouse TNF-α ELISA kit and the Mouse IL-1β ELISA kit respectively.

[0197] Table 11: Experimental design

[0198]

[0199]

[0200] Results: See Tables 12 and 13.

[0201] Table 12: Summary table of TNF-α data

[0202] Group Average concentration (pg / mL) SD p-value Blank control (BC) 325.746 17.748 / Negative control (NC) 573.633 17.568 <![CDATA[0.006 ## <!-- 17 -->]]> Positive control (PC) 373.352 8.721 <![CDATA[0.002 ** > CA-0.5% 355.452 13.453 <![CDATA[0.005 ** > CA-0.1% 474.860 6.577 <![CDATA[0.007 ** >

[0203] Table 13: Summary Table of IL-1β Data

[0204] 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 ** >

[0205] Note: When using the t-test two-tailed test method for statistical analysis, compared with the NC group and the BC group, significance is indicated by #, p-value < 0.05 is expressed as # , p-value < 0.01 is expressed as ## . When comparing the sample group, the PC group with the NC group, significance is indicated by *, p-value < 0.05 is expressed as *, p-value < 0.01 is expressed as **.

[0206] Conclusion:

[0207] Compared with the BC group, the secretion of macrophage inflammatory factor TNF-α in the NC group increased significantly (p < 0.01), indicating that the LPS-stimulated model was successfully established in this experiment. Compared with the NC group, at the administration concentration of 100 μg / mL of dexamethasone in the PC group, the secretion of macrophage inflammatory factor TNF-α decreased significantly (p < 0.01), indicating that the positive control was effective in this experiment. Compared with the NC group, when the sample was at the administration concentrations of 0.5% (m / V) and 0.1% (m / V), the secretion of macrophage inflammatory factor TNF-α decreased significantly (p < 0.01), indicating that the sample has anti-inflammatory, soothing and other effects.

[0208] Compared with the BC group, the secretion of macrophage inflammatory factor IL-1β in the NC group increased significantly (p < 0.01), indicating that the LPS-stimulated model was successfully established in this experiment. Compared with the NC group, at the administration concentration of 100 μg / mL of dexamethasone in the PC group, the secretion of macrophage inflammatory factor IL-1β decreased significantly (p < 0.01), indicating that the positive control was effective in this experiment. Compared with the NC group, when the sample was at the administration concentrations of 0.5% (m / V) and 0.1% (m / V), the secretion of macrophage inflammatory factor IL-1β decreased significantly (p < 0.01). Based on the LPS-stimulated macrophage anti-inflammatory model, when the sample was at the administration concentrations of 0.1% (m / V) and 0.5% (m / V), the secretion of macrophage inflammatory factors TNF-α and IL-1β both decreased significantly, showing a statistically significant difference compared with the NC group (p < 0.01), indicating that the sample has anti-inflammatory, soothing and other effects.

[0209] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes, appropriate alterations or combinations to 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 draw on the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and alterations are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A culture medium for culturing Centella asiatica stem cells with a high madecassoside content, characterized in that: The culture medium is based on the B5 liquid culture medium, and is supplemented with 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 based on the volume of the B5 culture medium. 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).

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