Culture method of rhodiola crenulata cell culture

Abstract

The embodiment of the specification provides a culture method of a rhodiola crenulata cell culture, which comprises the following steps: (a) extracting an explant of rhodiola crenulata, inoculating the explant into an MS solid culture medium containing 1-naphthylacetic acid and 6-benzyladenine, and inducing to form a callus; (b) screening out a target cell line through continuous culture; and (c) inoculating the target cell line into an improved MS liquid culture medium for culturing to obtain the target rhodiola crenulata cell culture.

Description

Technical Field

[0001] This specification relates to the technical field of plant cell culture, and particularly relates to a method for culturing Rhodiola crenulata cell cultures. Background Art

[0002] Rhodiola rosea L. is a perennial herbaceous plant growing in cold regions, mainly distributed in the high mountains and polar regions of Eurasia. Rhodiola contains various active ingredients such as salidroside, rhodiolin, tyrosol, tyrosol glycoside, etc., and has various pharmacological activities such as anti-fatigue, antioxidant, anti-inflammatory, antidepressant, and immune enhancement, and is widely used in the fields of medicine, health products, and cosmetics.

[0003] Traditional extraction methods of Rhodiola mainly rely on wild resources or artificial cultivation, and extract active ingredients through physical or chemical means. However, these methods have many problems: the growth cycle of Rhodiola is long, wild resources are scarce, and it cannot meet the growing market demand; over-collection of wild Rhodiola will damage the ecological environment; traditional extraction methods have low extraction efficiency and it is difficult to obtain high-purity active ingredients; the process of artificial cultivation and extraction is complex and the cost is high.

[0004] Cell culture technology is a method for obtaining plant secondary metabolites by culturing plant cells, tissues or organs in vitro. Compared with traditional plant extraction methods, cell culture technology has the following advantages: (1) Sustainability: Through in vitro culture technology, it is possible to achieve continuous production of plant cells without relying on natural resources; (2) High efficiency: Cell culture can produce a large number of plant cells in a short time with high extraction efficiency; (3) Environmentally friendly: The cell culture process is environmentally friendly and does not damage the ecosystem; (4) Stability: By optimizing the culture conditions, it is possible to stably produce high-quality plant cells and secondary metabolites. In recent years, researchers have begun to pay attention to the culture of Rhodiola cells and the production of secondary metabolites. However, current research mainly focuses on laboratory-scale culture, and there are the following problems: the culture conditions are unstable, the culture conditions of Rhodiola cells are complex, and it is difficult to maintain stability in large-scale production; the composition of the culture medium has not been optimized, and the composition and formula of the culture medium used in existing research are not unified, resulting in low efficiency of cell growth and metabolite production; there is a lack of large-scale production processes, and current research mostly stays at the laboratory scale, lacking a systematic large-scale production process and unable to meet market demand.

[0005] In view of this, in order to overcome the limitations of traditional extraction methods and the deficiencies of existing cell culture technologies, it is necessary to provide a method for culturing Rhodiola crenulata cell cultures, which can improve the quality and yield of Rhodiola cells by optimizing the cells themselves, the cell culture medium, the culture conditions and the bioreactor design, and provide an efficient and environmentally friendly raw material source for the fields of medicine, health products, and cosmetics. Summary of the Invention

[0006] One or more embodiments of the present specification provide a method for culturing Rhodiola crenulata cell cultures, comprising the following steps: (a) extracting explants of Rhodiola crenulata and inoculating them into an MS solid medium containing 1-naphthaleneacetic acid and 6-benzyladenine to induce the formation of callus; (b) screening out the target cell line through continuous culture; and (c) inoculating the target cell line into a modified MS liquid medium for culture to obtain the target Rhodiola crenulata cell cultures.

[0007] In some embodiments, step (a) further includes performing aseptic treatment on the explants, and the aseptic treatment includes: soaking in 70%-90% (v / v) ethanol for 20-40 s, soaking in 0.1%-0.2% (v / v) mercuric chloride (HgCl2) for 3-7 min, and rinsing with sterile water.

[0008] In some embodiments, in step (a), the concentration of 1-naphthaleneacetic acid is 1-5 mg / L, and the concentration of 6-benzyladenine is 0.3-0.7 mg / L.

[0009] In some embodiments, in step (a), the culture conditions for inducing the formation of callus include: relative humidity of 73%-77%, temperature of 23°C-27°C, light cycle of 10-14 hours per day, and light intensity of 1000-3000 lux.

[0010] In some embodiments, in step (b), the continuous culture is 18-22 subcultures.

[0011] In some embodiments, in step (c), the modified MS liquid medium contains 1-naphthaleneacetic acid and kinetin, the concentration of 1-naphthaleneacetic acid in the modified MS liquid medium is 0.3-0.7 mg / L, and the concentration of kinetin is 0.5-1.5 mg / L.

[0012] In some embodiments, in step (c), the modified MS liquid medium includes: 2000-2500 mg / L potassium nitrate (KNO 3 ), 110-150 mg / L ammonium nitrate (NH 4 NO 3 ), 75-90 mg / L potassium dihydrogen phosphate (KH 2 PO 4 ), 160-180 mg / L magnesium sulfate (MgSO 4 ·7H 2 O), 190-210 mg / L calcium chloride (CaCl 2 ·2H 2 O).

[0013] In some embodiments, step (c) further includes subjecting the target cell line to at least one of cryopreservation treatment and ultraviolet radiation treatment to further induce the accumulation of effective secondary metabolites in the Rhodiola crenulata cell culture, thereby obtaining the target Rhodiola crenulata cell culture.

[0014] In some embodiments, the cryopreservation treatment is carried out for 12 - 24 h at a temperature of 3 - 5 °C; the ultraviolet radiation treatment is carried out for 6 - 24 h at a light intensity of 0.5 - 2.5 W / m 2 , and the wavelength is 260 - 280 nm.

[0015] One or more embodiments of the present specification provide a Rhodiola crenulata cell culture, which is prepared by the method as described above. The contents of effective secondary metabolites in the dried product of the Rhodiola crenulata cell culture are: salidroside 0.58% - 7.8%, icariside D2 1.2% - 9.6%, tyrosol 0.3% - 3.2%, total flavonoids 0.5% - 3.1%, and total polyphenols 0.9% - 4.1%.

[0016] In some embodiments, the expressions of phenylalanine ammonia-lyase (PAL), tyrosine decarboxylase (TyDc), and uridine diphosphate glucose transferase (UDPGT) genes in the Rhodiola crenulata cell culture are up-regulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present specification will be further described by way of exemplary embodiments, which will be described in detail through the accompanying drawings. These embodiments are not restrictive, where:

[0018] Figure 1 is a schematic diagram of callus of four forms of Rhodiola crenulata according to some embodiments of the present specification (A: yellow Rhodiola crenulata callus; B: green Rhodiola crenulata callus; C: red Rhodiola crenulata callus; D: purple Rhodiola crenulata callus; E: biomass accumulation of callus of four forms in one subculture cycle);

[0019] Figure 2 is a schematic diagram of the analysis of the expression levels of PAL1, TyDC, and UDPGT genes in the cells of green, red, and purple Rhodiola crenulata according to some embodiments of the present specification (A: PAL1; B: TyDC; C: UDPGT);

[0020] Figure 3 is a schematic diagram of the difference in salidroside content in the Rhodiola crenulata cell culture under conventional MS medium and modified MS culture conditions according to some embodiments of the present specification;

[0021] Figure 4 Schematic diagram showing the changes in the expression levels of the key genes TyDC and UDPGT for salidroside synthesis under different stress conditions as shown in some embodiments of this specification (A: TyDC; B: UDPGT);

[0022] Figure 5 Schematic diagram for comparing the HPLC fingerprint of the cell culture of Rhodiola crenulata and the standard medicinal material as shown in some embodiments of this specification;

[0023] Figure 6 Schematic diagram showing the differences in the ability of cell cultures of different Rhodiola plants to synthesize salidroside as shown in some embodiments of this specification. Detailed implementation manners

[0024] To more clearly illustrate the technical solutions of the embodiments of this specification, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings.

[0025] The embodiments of this specification provide a method for culturing the cell culture of Rhodiola crenulata, including the following steps: (a) Extracting the explants of Rhodiola crenulata and inoculating them into an MS solid medium containing 1-naphthylacetic acid and 6-benzyladenine to induce the formation of callus; (b) Screening out the target cell line through continuous culture; and (c) Inoculating the target cell line into a modified MS liquid medium for culture to obtain the target cell culture of Rhodiola crenulata.

[0026] Callus refers to a mass of undifferentiated and disorderly proliferating parenchyma cells formed by local cells through dedifferentiation (losing their original differentiation characteristics) and redivision when the plant body is mechanically damaged, pathogen-infected, or artificially induced. Callus is not only an important structural basis for plant self-repair and regeneration but also a key material for realizing cell totipotency and redifferentiation in plant tissue culture.

[0027] The explants of Rhodiola crenulata refer to the living tissue or organ fragments separated from the Rhodiola crenulata plant for tissue culture. In some embodiments, the explants of Rhodiola crenulata can be the stem tips, leaves, root segments, or buds of Rhodiola crenulata.

[0028] MS solid medium (Murashige and Skoog Solid Medium) is a basic medium widely used in plant tissue culture. Based on MS liquid medium, it is solidified by adding agar to form a solid medium suitable for the growth of plant cells, tissues or organs. MS medium contains rich nutrients such as inorganic salts, vitamins, amino acids and sucrose, which can meet the growth requirements of most plant cells. The high concentrations of nitrate and potassium ions in MS solid medium are suitable for promoting the division of plant cells and the formation of callus.

[0029] 1-Naphthaleneacetic acid (NAA) is a synthetic plant growth regulator that can mimic the physiological functions of natural auxins (such as indole-3-acetic acid, IAA), promoting plant cell elongation, division and tissue differentiation. NAA is often used to induce callus formation, promote root development and regulate the growth and development process of plants.

[0030] 6-Benzylaminopurine (6-BA) is a synthetic cytokinin-like plant growth regulator that can mimic the physiological functions of natural cytokinins. 6-BA mainly promotes the division and proliferation of plant cells, delays tissue senescence, and participates in regulating the growth and development process of plants. In plant tissue culture, 6-BA is often used to induce bud differentiation, promote lateral bud germination and improve the regeneration ability of callus. When used in combination with NAA, 6-BA can effectively regulate organogenesis and morphogenesis of plants.

[0031] In some embodiments, in step (a), the concentration of NAA can be 1-5 mg / L, and the concentration of 6-BA can be 0.3-0.7 mg / L.

[0032] In some embodiments, in step (a), the concentration of NAA can be 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, or 5 mg / L. In some embodiments, in step (a), the concentration of NAA can be 3 mg / L.

[0033] In some embodiments, in step (a), the concentration of 6-BA can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, or 0.7 mg / L. In some embodiments, in step (a), the concentration of 6-BA can be 0.5 mg / L.

[0034] In some embodiments, in step (a), the concentration of NAA is 3 mg / L and the concentration of 6-BA is 0.5 mg / L.

[0035] In some embodiments, step (a) further includes performing aseptic treatment on the explant, and the aseptic treatment can be achieved in various ways. In some embodiments, in step (a), performing aseptic treatment on the explant may include: soaking in 70%-90% (v / v) ethanol for 20-40 s, soaking in 0.1%-0.2% (v / v) mercury chloride (HgCl2) for 3-7 min, and rinsing with sterile water.

[0036] In some embodiments, the concentration of ethanol can be 70%, 75%, 80%, 85%, or 90%. In some embodiments, the concentration of ethanol can be 75%.

[0037] In some embodiments, the soaking time of ethanol can be 20 s, 25 s, 30 s, 35 s, or 40 s. In some embodiments, the soaking time of ethanol can be 30 s.

[0038] In some embodiments, the concentration of mercury chloride can be 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, or 0.2%. In some embodiments, the concentration of mercury chloride can be 0.1%.

[0039] In some embodiments, the soaking time of mercury chloride can be 3 min, 4 min, 5 min, 6 min, or 7 min. In some embodiments, the soaking time of mercury chloride can be 5 min.

[0040] In some embodiments, the number of times of rinsing with sterile water can be 5-6 times.

[0041] In some embodiments, in step (a), performing aseptic treatment on the explant includes: soaking in 75% (v / v) ethanol for 30 s, soaking in 0.1% (v / v) mercury chloride (HgCl2) for 5 min, and rinsing with sterile water.

[0042] In some embodiments, in step (a), the culture conditions for inducing the formation of callus include: relative humidity of 73%-77%, temperature of 23°C-27°C, light cycle of 10-14 hours per day, and light intensity of 1000-3000 lux.

[0043] Relative humidity refers to the ratio of the actual water vapor content in the air to the saturated water vapor content at a certain temperature, usually expressed as a percentage (%). Relative humidity reflects the humidity of the air and is an important indicator for measuring environmental humidity.

[0044] In some embodiments, in the culture conditions for inducing the formation of callus, the relative humidity can be 73%, 74%, 75%, 76%, or 77%. In some embodiments, in the culture conditions for inducing the formation of callus, the relative humidity can be 75%.

[0045] In some embodiments, in the culture conditions for inducing callus formation, the temperature can be 23°C, 24°C, 25°C, 26°C, or 27°C. In some embodiments, in the culture conditions for inducing callus formation, the temperature can be 25°C.

[0046] In some embodiments, in the culture conditions for inducing callus formation, the light cycle can be 10 hours / day, 11 hours / day, 12 hours / day, 13 hours / day, or 14 hours / day. In some embodiments, in the culture conditions for inducing callus formation, the light cycle can be 12 hours / day.

[0047] In some embodiments, in the culture conditions for inducing callus formation, the light intensity can be 1000 lux, 1500 lux, 2000 lux, 2500 lux, or 3000 lux. In some embodiments, in the culture conditions for inducing callus formation, the light intensity can be 2000 lux.

[0048] In some embodiments, in step (a), the culture conditions for inducing callus formation include: relative humidity of 75%, temperature of 25°C, light cycle of 12 hours / day, and light intensity of 1000 - 3000 lux.

[0049] In callus culture, continuous culture refers to the process of subculturing callus multiple times in a suitable medium and conditions to maintain its continuous growth and proliferation. The purpose of continuous culture is to maintain the activity and undifferentiated state of callus, prevent its aging or spontaneous differentiation, and at the same time provide sufficient biological materials for subsequent experiments or applications. Through continuous culture, not only can callus be preserved for a long time, but also target cell lines with strong growth and stable genetics can be screened.

[0050] The target cell line refers to the cell line obtained by screening callus after continuous culture, which has good growth potential, strong ability to accumulate metabolites, and stable gene expression levels of the key enzymes for salidroside synthesis.

[0051] In some embodiments, in step (b), the continuous culture can be 18 - 22 subcultures.

[0052] In some embodiments, in step (b), the continuous culture can be 18, 19, 20, 21, or 22 subcultures.

[0053] In some embodiments, in step (b), the continuous culture is 20 subcultures.

[0054] In some embodiments, in step (c), the modified MS liquid medium contains 1-naphthaleneacetic acid and kinetin. The concentration of 1-naphthaleneacetic acid in the modified MS liquid medium can be 0.3 - 0.7 mg / L, and the concentration of kinetin can be 0.5 - 1.5 mg / L.

[0055] Kinetin (KT) is a non-natural cytokinin that promotes cell division. KT acts synergistically with NAA to jointly regulate the growth and development process of plants.

[0056] In some embodiments, in step (c), the concentration of NAA in the modified MS liquid medium can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, or 0.7 mg / L. In some embodiments, in step (c), the concentration of NAA in the modified MS liquid medium can be 0.5 mg / L.

[0057] In some embodiments, in step (c), the concentration of kinetin in the modified MS liquid medium can be 0.5 mg / L, 0.75 mg / L, 1.0 mg / L, 1.25 mg / L, or 1.5 mg / L. In some embodiments, in step (c), the concentration of kinetin in the modified MS liquid medium can be 1.0 mg / L.

[0058] In some embodiments, in step (c), the modified MS liquid medium contains 1-naphthaleneacetic acid and kinetin. The concentration of 1-naphthaleneacetic acid in the modified MS liquid medium is 0.5 mg / L, and the concentration of kinetin is 1.0 mg / L.

[0059] In some embodiments, in step (c), the modified MS liquid medium may include: 2000 - 2500 mg / L potassium nitrate (KNO 3 ), 110 - 150 mg / L ammonium nitrate (NH 4 NO 3 ), 75 - 90 mg / L potassium dihydrogen phosphate (KH 2 PO 4 ), 160 - 180 mg / L magnesium sulfate (MgSO 4 ·7H 2 O), 190 - 210 mg / L calcium chloride (CaCl 2 ·2H 2 O).

[0060] In some embodiments, the concentration of potassium nitrate (KNO 3 ) in the modified MS liquid medium can be 2000 mg / L, 2100 mg / L, 2200 mg / L, 2300 mg / L, 2400 mg / L, or 2500 mg / L.

[0061] In some embodiments, the concentration of ammonium nitrate (NH 4 NO 3 ) in the modified MS liquid medium can be 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, or 150 mg / L.

[0062] In some embodiments, the concentration of potassium dihydrogen phosphate (KH 2 PO 4 ) in the modified MS liquid medium can be 75 mg / L, 80 mg / L, 85 mg / L, or 90 mg / L.

[0063] In some embodiments, the concentration of magnesium sulfate (MgSO 4 ·7H 2 O) in the modified MS liquid medium can be 160 mg / L, 165 mg / L, 170 mg / L, 175 mg / L, or 180 mg / L.

[0064] In some embodiments, the concentration of calcium chloride (CaCl 2 ·2H 2 O) in the modified MS liquid medium can be 190 mg / L, 195 mg / L, 200 mg / L, 205 mg / L, or 210 mg / L.

[0065] In some embodiments, step (c) further includes subjecting the target cell line to at least one of cryopreservation treatment and ultraviolet radiation treatment to further induce the accumulation of effective secondary metabolites in the Rhodiola crenulata cell culture, thereby obtaining the target Rhodiola crenulata cell culture.

[0066] In some embodiments, the time of cryopreservation treatment can be 12 - 24 h, and the temperature can be 3 - 5 °C; the time of ultraviolet radiation treatment can be 6 - 24 h, and the light intensity can be 0.5 - 2.5 W / m 2 , and the wavelength can be 260 - 280 nm.

[0067] In some embodiments, the time of cryopreservation treatment can be 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, or 24 h.

[0068] In some embodiments, the temperature of cryopreservation treatment can be 3 °C, 3.5 °C, 4 °C, 4.5 °C, or 5 °C.

[0069] In some embodiments, the time of ultraviolet radiation treatment can be 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, or 24 h.

[0070] In some embodiments, the light intensity of the ultraviolet radiation treatment can be 0.5 W / m 2 、1 W / m 2 、1.5 W / m 2 、2 W / m 2 、or 2.5 W / m 2 。

[0071] In some embodiments, the wavelength of the ultraviolet radiation treatment can be 260 nm, 265 nm, 270 nm, 275 nm, or 280 nm.

[0072] The embodiments of this specification provide a Rhodiola crenulata cell culture, which is prepared by the above method. The content of effective secondary metabolites in the dried product of the Rhodiola crenulata cell culture is: salidroside 0.58% - 7.8%, icariside D2 1.2% - 9.6%, tyrosol 0.3% - 3.2%, total flavonoids 0.5% - 3.1%, and total polyphenols 0.9% - 4.1%.

[0073] Salidroside is an important bioactive ingredient in plants of the genus Rhodiola such as Rhodiola crenulata, and belongs to phenyl ethanol glycoside compounds. Salidroside is one of the main medicinal components of Rhodiola, and has a variety of significant pharmacological effects, including antioxidant, anti-fatigue, anti-inflammatory, anti-tumor, anti-hypoxia, and immune-enhancing effects. Salidroside can help the body cope with environmental stress and improve adaptability by scavenging free radicals, regulating cellular energy metabolism, and protecting mitochondrial function. In the fields of medicine and health products, salidroside is widely used to improve physical fatigue, relieve neurasthenia, enhance cardiovascular function, and assist in the treatment of depression, etc.

[0074] Icariside D2 is a secondary metabolite in Rhodiola crenulata, and belongs to flavonoid glycoside compounds. Icariside D2 is a derivative of icariin and has a variety of biological activities, including antioxidant, anti-inflammatory, anti-tumor, and neuroprotective effects. Research shows that icariside D2 can inhibit the proliferation of tumor cells and induce their apoptosis by regulating cell signaling pathways (such as the PI3K / AKT and MAPK pathways), and at the same time, it also has the potential to improve cardiovascular function and enhance immunity.

[0075] Tyrosol is a natural phenolic compound in Rhodiola crenulata and is also one of its important bioactive components. Tyrosol is a simple water-soluble phenolic substance with significant antioxidant, anti-inflammatory, anti-aging, and neuroprotective effects. Tyrosol can help the body resist external environmental stress and delay cell aging by scavenging free radicals, inhibiting oxidative stress reactions, and protecting the integrity of cell membranes. In addition, tyrosol has also been shown to have cardiovascular protective effects, improving blood circulation and reducing the risk of atherosclerosis.

[0076] In some embodiments, the contents of salidroside and tyrosol can be determined by high performance liquid chromatography (HPLC). The specific method is as follows: methanol-water (the volume ratio can be 15:85) is used as the mobile phase, the flow rate can be 1.0 mL / min, the detection wavelength can be 275 nm, and the column temperature can be 20 °C.

[0077] In some embodiments, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF) can be used to identify other secondary metabolite components. In some embodiments, other secondary metabolite components may include salidroside isomers, derivatives, and other metabolites.

[0078] In some embodiments, the expressions of phenylalanine ammonia-lyase (PAL), tyrosine decarboxylase (TyDc), and uridine diphosphate glucose transferase (UDPGT) genes in Rhodiola crenulata cell cultures are up-regulated.

[0079] In some embodiments, phenylalanine ammonia-lyase (PAL), tyrosine decarboxylase (TyDc), and uridine diphosphate glucose transferase (UDPGT) in Rhodiola crenulata cell cultures are key enzymes for salidroside synthesis, and their gene expression levels can be measured in various ways. In some embodiments, the gene expression levels of PAL, TyDc, and UDPGT can be measured by quantitative real-time PCR (qPCR).

[0080] Quantitative Real-time PCR (qPCR) is a highly sensitive and specific technique for detecting gene expression levels. qPCR quantitatively analyzes the initial template amount of the target gene by monitoring the accumulation of fluorescence signals during the PCR amplification process, thereby accurately determining the gene expression level.

[0081] Phenylalanine Ammonia-Lyase (PAL) is one of the key enzymes in the biosynthesis of salidroside, catalyzing the conversion of phenylalanine to cinnamic acid and serving as the starting enzyme in the phenylpropanoid metabolic pathway. The activity of PAL affects the synthesis efficiency of phenyl ethanol glycosides such as salidroside. The expression and activity of PAL enzyme are regulated by various factors, including light, temperature, hormones, and external stresses. In the biosynthesis of salidroside, the reaction catalyzed by PAL is one of the key steps in generating the precursor tyrosol. By regulating the expression of the PAL gene or enzyme activity, the yield of salidroside can be significantly increased.

[0082] Tyrosine Decarboxylase (TyDc) is one of the key enzymes in the biosynthetic pathway of salidroside, responsible for catalyzing the decarboxylation of tyrosine to form tyramine, which is an important precursor step in the synthesis of salidroside. The activity of TyDC affects the synthesis efficiency of salidroside, and its expression level is regulated by various internal and external factors, including hormones, light, and external stresses.

[0083] Uridine Diphosphate Glucose Transferase (UDPGT) is a key glycosyltransferase that catalyzes the glycosylation reaction of tyrosol to produce salidroside in the biosynthesis of salidroside. By regulating the activity of UDPGT, the synthesis efficiency of salidroside can be optimized.

[0084] This specification example also provides the synthesis of salidroside in different Rhodiola cell lines.

[0085] In some embodiments, different Rhodiola species may include Rhodiola sacra, Rhodiola sachalinensis, and Rhodiola rosea. In some embodiments, the salidroside synthesis ability of Rhodiola sacra, Rhodiola sachalinensis, and Rhodiola rosea is similar to that of Rhodiola crenulata.

[0086] In some embodiments of this specification, by optimizing the culture medium and stress treatment conditions, the large-scale synthesis of salidroside is achieved. Through continuous culture and screening, a cell line with high-efficiency synthesis of salidroside is obtained, significantly improving the production efficiency of salidroside and providing reliable technical support for large-scale industrial production. Example Example 1: Induction of Rhodiola crenulata Callus

[0087] Extract explants from healthy Rhodiola crenulata plants. Use sterile tools to cut young shoot tips or leaves. Under sterile conditions, soak them in 75% (v / v) alcohol for 30 s, rinse with sterile water 5 - 6 times, dry with filter paper, and then soak in 0.1% (v / v) mercuric chloride for 5 min, rinse with sterile water 5 - 6 times, and dry with filter paper to ensure the extracted tissue blocks are sterile.

[0088] Under the laminar flow hood, use a scalpel to cut the above - sterilized explant materials into 0.5 cm×0.5 cm squares. Using MS medium as the basal medium, add 3 mg / L 1 - naphthylacetic acid (NAA) and 0.5 mg / L 6 - benzyladenine (6 - BA) as growth regulators to induce callus of the explants. The carbon source in the medium is 3% (w / v) sucrose, and additionally add 0.1 g / L L - ascorbic acid (vitamin C, VC) to prevent browning during the induction of explants. Add 0.8% (w / v) agar to the medium to prepare MS solid medium.

[0089] The culture conditions during the induction of Rhodiola crenulata callus are as follows: the relative humidity is 73% - 77%, cultured under a constant temperature of 23℃ - 27℃, the light cycle is 12 hours per day, and the light intensity is 1000 - 3000 lux.

[0090] During the induction period, use a sterile scalpel to damage the explants every 1 week to stimulate the callusing of the explants. After 8 weeks of induction, select the significantly callused explants for sub - culture. Example 2: Screening of Rhodiola crenulata target cell lines

[0091] During the solid - state culture of Rhodiola crenulata cells, screening high - quality cell clumps is a key step for cell expansion and large - scale production. High - quality cell clumps have good growth potential and high metabolite accumulation ability. The formula of the sub - culture medium is: based on MS medium, add 0.5 mg / L NAA and 1 mg / L kinetin (KT), the carbon source is 3% (w / v) sucrose, and 0.6% (w / v) agar. Taking 3 weeks as one sub - culture cycle, sub - culture 100 bottles each time, and screen out cell clumps with a significant increase in volume, regular shape, smooth edges and uniformity.

[0092] As Figure 1 shown, conduct 20 successive passages of culture, and screen out 4 types of callus with stable states. Distinguished by color, they are yellow (A), green (B), red (C) and purple (D) Rhodiola crenulata callus.

[0093] Statistically analyze the biomass accumulation of different - shaped callus in one sub - culture cycle (as Figure 1(as shown in (E)), the initial inoculation amount was 1 g / bottle. The results showed that the biomass of the callus of Rhodiola crenulata with large yellow flowers accumulated 1.3 times, the biomass of the green callus accumulated 6.7 times, the biomass of the red callus accumulated 5.4 times, while the biomass of the purple callus accumulated 10.5 times.

[0094] In view of the slow biomass accumulation of the yellow cell line, which did not meet the actual production requirements, no further exploration was carried out in the subsequent examples. Further, the real-time fluorescence quantitative PCR technology (qPCR) was used to analyze the gene expression levels of the key enzymes encoding the salidroside synthesis pathway in the green, red, and purple Rhodiola crenulata cell lines. As Figure 2 shown, the results indicated that in the purple Rhodiola crenulata cell line, the expression levels of the key enzymes phenylalanine ammonia-lyase PAL, tyrosine decarboxylase TyDc, and uridine diphosphate glucose transferase UDPGT in the mRNA level were extremely low. The above three key enzymes could be expressed in the green Rhodiola crenulata cell line, while the expression level of the key enzyme TyDc was relatively low in the red Rhodiola crenulata cell line. Considering comprehensively, the green Rhodiola crenulata cell line was selected as the target cell line for subsequent process screening. Table 1 Example 3: Screening of the salidroside induction process for Rhodiola crenulata cell cultures suitable for production

[0095] The solid-cultured green Rhodiola crenulata cell line was inoculated into the MS liquid medium containing 0.5 mg / L NAA, 1 mg / L KT, and 3% (w / v) sucrose. The pH of the medium was 5.8 - 6.0, and it was cultured with shaking at a speed of 120 rpm, maintaining the temperature at 23°C - 27°C, the light cycle was 16 hours of light / 8 hours of darkness, and the light intensity was 1000 ± 100 lux.

[0096] With a subculture period of 21 days, after subculturing 3 times, a suspension cell line with uniform state was obtained.

[0097] The composition of the substances in the modified MS medium is shown in Table 2, where the concentration range of potassium nitrate (KNO 3 ) in the macronutrients is 2000 - 2500 mg / L; the concentration range of ammonium nitrate (NH 4 NO 3 ) is 110 - 150 mg / L; the concentration range of potassium dihydrogen phosphate (KH 2 PO 4 ) is 75 - 90 mg / L; the concentration range of magnesium sulfate (MgSO 4 ·7H 2 O) is 160 - 180 mg / L; the concentration range of calcium chloride (CaCl 2 ·2H2 The concentration range of O) is 190 - 210 mg / L. The other trace element components, iron salts and organic components are the same as those in the conventional MS medium. Table 2

[0098] As Figure 3 shown, by comparing the differences in the salidroside synthesis ability of Rhodiola crenulata cell cultures cultured in the conventional MS medium and the modified MS medium, the results show that the salidroside synthesis ability of Rhodiola crenulata in the modified MS medium has increased by 2.33 times.

[0099] Furthermore, the modified MS medium was used as the medium for subsequent Rhodiola crenulata cell cultures, and on this basis, the optimal induction conditions for salidroside synthesis were screened. Take the suspended cells of Rhodiola crenulata cultured for 14 days, set different conditions to treat the suspended cells, and use qPCR technology to detect the expression levels of the genes encoding the key enzymes for salidroside synthesis.

[0100] The candidate induction conditions were set to treat the cells with 4°C freezing treatment (Cold), 300 mM D-mannitol treatment (Mannitol), 100 mM NaCl treatment, 5% sucrose treatment (Sucrose), ultraviolet radiation treatment (UV), acid treatment (pH 4.5) and alkali treatment (pH 10.0) for 12 hours, extract total RNA, reverse transcribe it into cDNA, and then perform qPCR detection to detect the changes in the expression levels of the genes encoding tyrosine decarboxylase (TyDc) and uridine diphosphate glucose transferase (UDPGT).

[0101] The detection results are as Figure 4 shown. Compared with the control group (Control), the fold differences in the relative expression levels of TyDc under the above treatment conditions are 3.8-fold, 1.2-fold, 0.8-fold, 0.8-fold, 0.9-fold, 0.5-fold and 0.7-fold respectively; compared with the control group (Control), the fold differences in the expression of UDPGT under the above treatment conditions are 1.2-fold, 1.2-fold, 2.2-fold, 1.5-fold, 3.6-fold, 1.1-fold and 0.01-fold respectively.

[0102] Based on the above results, perform 4°C freezing treatment and UV ultraviolet radiation (0.5 - 2.5 W / m 2, the suspension cells of Rhodiola crenulata after subculture and growing for 14 days were treated with ultraviolet light (wavelength range: 260 - 280 nm), and the synthesis of salidroside was detected by high performance liquid chromatography (HPLC). The results are shown in Table 3. After the suspension cells of Rhodiola crenulata were frozen at 4°C for 24 hours and then irradiated with ultraviolet light for 12 hours (HJT - 13), the content of salidroside in the dried product was the highest, reaching 77.8 mg / g. Table 3 Example 4: Characterization of Active Secondary Metabolites in Cell Cultures of Rhodiola crenulata

[0103] According to the optimal process scheme in Example 3, the suspension cells of Rhodiola crenulata after subculture and growing for 14 days were frozen at 4°C for 24 hours, then irradiated with ultraviolet light for 12 hours, and then the culture was collected and dried in an oven at 55°C for 12 hours.

[0104] According to the detection method of salidroside in Rhodiola crenulata in the Pharmacopoeia of the People's Republic of China (2020 Edition), the contents of salidroside and tyrosol were determined by HPLC. The mobile phase was methanol - water (volume ratio 15:85), the flow rate was 1.0 mL / min, the detection wavelength was 275 nm, and the column temperature was 20°C. A standard curve was established, and the contents of salidroside and tyrosol were calculated by the peak area.

[0105] As Figure 5 shown, by comparing the HPLC fingerprint spectra of the reference substances (1: salidroside; 2: tyrosol), cell cultures of Rhodiola crenulata, and standard medicinal materials of Rhodiola crenulata, the similarity between the HPLC fingerprint spectra of cell cultures of Rhodiola crenulata and standard medicinal materials of Rhodiola crenulata was above 90%, and the characteristic peaks of the two standard compounds, salidroside and tyrosol, could be detected in both.

[0106] Furthermore, in order to determine the components of other characteristic peaks in cell cultures of Rhodiola crenulata, UPLC - Q - TOF technology was used for metabolite component identification. In addition to salidroside and tyrosol, isorhynchophyllin D2, an isomer of salidroside, methyl salidroside, a derivative of salidroside, and methyl salidroside, as well as protocatechuic acid, were also identified.

[0107] Furthermore, according to the optimal process scheme in Example 3, 10 batches of Rhodiola crenulata cell cultures were cultivated, and the content fluctuation ranges of the main metabolites in the cultures of different batches were detected. The statistical results are shown in Table 4. The average content of salidroside in the dried Rhodiola crenulata cell cultures was 3.89%, with the content ranging from 0.58% to 7.8%; the average content of icariside D2 was 4.86%, with the content ranging from 1.2% to 9.6%; the average content of tyrosol was 1.3%, with the content ranging from 0.3% to 3.2%; the average content of total flavonoids was 1.92%, with the content ranging from 0.5% to 3.1%; the average content of total polyphenols was 2.72%, with the content ranging from 0.9% to 4.1%. Table 4 Example 5: Process for inducing the production of salidroside in Rhodiola sacra, Rhodiola cretinii and Rhodiola rosea

[0108] Explants were extracted from healthy Rhodiola sacra, Rhodiola cretinii and Rhodiola rosea plants, and MS solid medium supplemented with 3 mg / L NAA and 0.5 mg / L 6-BA was used for callus induction of the explants.

[0109] Screening of the target cell line was carried out on the solid medium, and cell clumps with good growth potential and high metabolite accumulation ability were selected.

[0110] After the selected target cell line was grown on the modified MS medium (the medium components were the same as in Example 3, containing 0.5 mg / L NAA and 1 mg / L KT, and the carbon source was 3% (w / v) sucrose) for 21 days, phenylalanine was used as the precursor substance, and the gene expression level of the key enzyme TyDc was induced to increase by freezing treatment (4 °C, 24 hours), and then the gene expression level of the key enzyme UDPGT was further induced to increase by ultraviolet radiation (12 hours).

[0111] The contents of salidroside and tyrosol were determined by HPLC. The mobile phase was methanol-water (volume ratio 15:85), the flow rate was 1.0 mL / min, the detection wavelength was 275 nm, and the column temperature was 20 °C. A standard curve was established, and the contents of salidroside and tyrosol were calculated by the peak area.

[0112] Compare the salidroside synthesis abilities in the cell cultures of Rhodiola crenulata, Rhodiola sacra, Rhodiola cretinii and Rhodiola rosea. The results are as Figure 6 shown. The statistical results indicate that there is no significant difference in the ability of the cell lines of the four Rhodiola species to produce salidroside after induction.

[0113] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification, so such modifications, improvements, and corrections still fall within the spirit and scope of the exemplary embodiments of this specification.

[0114] At the same time, this specification uses specific terms to describe the embodiments of this specification. Such as "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that the "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0115] In addition, unless explicitly stated in the claims, the order of the processing elements and sequences, the use of numerical letters, or the use of other names in this specification are not used to limit the order of the processes and methods of this specification. Although some currently considered useful embodiments of the invention are discussed through various examples in the above disclosure, it should be understood that such details only serve the purpose of illustration. The appended claims are not limited to the disclosed embodiments. On the contrary, the claims are intended to cover all modifications and equivalent combinations that conform to the essence and scope of the embodiments of this specification. For example, although the system components described above can be implemented by hardware devices, they can also be implemented only through software solutions, such as installing the described system on existing servers or mobile devices.

[0116] Similarly, it should be noted that, in order to simplify the expression of the disclosure of this specification and thus help the understanding of one or more embodiments of the invention, in the previous description of the embodiments of this specification, sometimes multiple features are merged into one embodiment, drawing, or its description. However, this disclosure method does not mean that the features required by the object of this specification are more than those mentioned in the claims. In fact, the features of the embodiments are less than all the features of the individual embodiments disclosed above.

[0117] In some embodiments, numbers are used to describe components and attribute quantities. It should be understood that such numbers used in the description of embodiments are, in some examples, modified by the modifiers "about", "approximately", or "substantially". Unless otherwise specified, "about", "approximately", or "substantially" indicate that the stated number allows for a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may vary according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should consider the specified significant digits and adopt the method of retaining the general number of digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, such numerical settings are as precise as possible within the feasible range.

[0118] For each patent, patent application, patent application publication, and other materials cited in this specification, such as articles, books, specifications, publications, documents, etc., their entire contents are hereby incorporated into this specification by reference. This excludes the application history files that are inconsistent with or conflict with the content of this specification, as well as the files that limit the broadest scope of the claims of this specification (currently or subsequently appended to this specification). It should be noted that if there are any inconsistencies or conflicts between the descriptions, definitions, and / or uses of terms in the attached materials of this specification and the content described in this specification, the descriptions, definitions, and / or uses of terms in this specification shall prevail.

[0119] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A method for culturing a Rhodiola rosea cell culture, characterized in that: The following steps are involved: (a) extracting explants of Rhodiola rosea and inoculating them into MS solid medium containing 1-naphthaleneacetic acid and 6-benzyladenine to induce callus formation; (b) screening out target cell lines through continuous culture; and (c) inoculating the target cell line into a modified MS liquid culture medium for culturing to obtain a target Rhodiola rosea cell culture.

2. The method according to claim 1, characterized in that Step (a) also includes aseptic treatment of the explant, which includes: soaking in 70%-90% (v / v) ethanol for 20-40s, soaking in 0.1%-0.2% (v / v) mercuric chloride (HgCl2) for 3-7min, and rinsing with sterile water.

3. The method according to claim 1, characterized in that In step (a), the concentration of the 1-naphthylacetic acid is 1-5 mg / L, and the concentration of the 6-benzyladenine is 0.3-0.7 mg / L.

4. The method according to claim 1, characterized in that: In step (a), the culture conditions for inducing callus formation include: relative humidity of 73%-77%, temperature of 23°C-27°C, photoperiod of 10-14 hours / day, and light intensity of 1000-3000 lux.

5. The method according to claim 1, characterized in that In step (b), the continuous culture is 18-22 generations of culture.

6. The method according to claim 1, characterized in that In step (c), the modified MS liquid culture medium contains 1-naphthylacetic acid and kinetin, the concentration of the 1-naphthylacetic acid in the modified MS liquid culture medium is 0.3-0.7 mg / L, and the concentration of the kinetin is 0.5-1.5 mg / L.

7. The method according to claim 1, characterized in that In step (c), the improved MS liquid culture medium includes: 2000-2500 mg / L potassium nitrate (KNO3), 110-150 mg / L ammonium nitrate (NH4NO3), 75-90 mg / L potassium dihydrogen phosphate (KH2PO4), 160-180 mg / L magnesium sulfate (MgSO4·7H2O), and 190-210 mg / L calcium chloride (CaCl2·2H2O).

8. The method according to claim 1, characterized in that Step (c) also includes subjecting the target cell line to at least one of low-temperature freezing treatment and ultraviolet radiation treatment to further induce the accumulation of effective secondary metabolites in the Rhodiola rosea cell culture to obtain the target Rhodiola rosea cell culture.

9. The method according to claim 8, characterized in that The low temperature freezing treatment lasts for 12-24 hours at a temperature of 3-5°C; the ultraviolet radiation treatment lasts for 6-24 hours at a light intensity of 0.5-2.5 W / m 2 , wavelength is 260-280nm.

10. A Rhodiola rosea cell culture, characterized in that: The Rhodiola rosea cell culture is prepared by the method described in any one of claims 1 to 9, and the content of effective secondary metabolites in the dried product of the Rhodiola rosea cell culture is: 0.58%-7.8% salidroside, 1.2%-9.6% icariin D2, 0.3%-3.2% tyrosol, 0.5%-3.1% total flavonoids, and 0.9%-4.1% total polyphenols.

11. The Rhodiola rosea cell culture according to claim 10, characterized in that The expression of phenylalanine ammonia lyase (PAL), tyrosine decarboxylase (TyDc) and uridine diphosphate glucose transferase (UDPGT) genes in the Rhodiola rosea cell culture was upregulated.

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