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Abstract

The present application belongs to the technical field of traditional Chinese medicine, and discloses a method for inducing Rehmannia glutinosa stem cells to produce terpenoids. The method is as follows: preparing a chitosan oligosaccharide solution; adding auxin and cytokinin to a basic MS medium to prepare a first modified MS medium; adjusting the concentrations of nitrogen source, phosphorus source and potassium source in the first modified MS medium, and adding plant peptides and β-cyclodextrin to prepare a second modified MS medium; inoculating Rehmannia glutinosa stem cells in the first modified MS medium and culturing to the logarithmic growth phase; then soaking the cells in the chitosan oligosaccharide solution for 0.5-5 hours; then transferring the cells to the second modified MS medium and culturing under illumination; after the culture ends, collecting the Rehmannia glutinosa stem cells and extracting terpenoids. The method provided by the present application can significantly improve the yield of terpenoids, optimize the purity, shorten the production cycle, and reduce the cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of traditional Chinese medicine, and specifically relates to a method for inducing Rehmannia glutinosa stem cells to produce terpenoids. BACKGROUND

[0002] Rehmannia glutinosa plays a crucial role in the long history of traditional Chinese medicine, and its medicinal value has been widely recognized and applied. Among them, iridoid compounds are the core components of Rehmannia glutinosa that exert numerous pharmacological effects. They exhibit significant activity in antioxidant, anti-inflammatory, neuroprotective, and immune regulation, and have an irreplaceable role in treating various diseases and maintaining human health.

[0003] For a long time, the industrial production of iridoid compounds has mainly relied on direct extraction from Rehmannia glutinosa plants. However, this traditional extraction method faces many severe challenges. On the one hand, Rehmannia glutinosa is strongly influenced by various complex environmental factors during natural growth. Different soil textures, fertility levels, pH levels, and variable climate conditions, including temperature, humidity, light duration, and intensity, all cause significant differences in the growth conditions and secondary metabolite accumulation of Rehmannia glutinosa plants. This makes the content of iridoid compounds extremely unstable, making it difficult to ensure stable yield supply. On the other hand, the growth cycle of Rehmannia glutinosa is relatively long, and it takes a lot of time and land resources from planting to harvesting, which undoubtedly increases the production and time costs, severely limiting its large-scale and efficient industrial production.

[0004] With the advent of modern biotechnology, the rise of plant tissue culture technology has brought light to this dilemma. In particular, the emergence of plant stem cell technology has made it possible to produce medicinal plant secondary metabolites in a controllable in vitro environment. Rehmannia glutinosa cambium meristem stem cells, with their high self-renewal ability and multi-directional differentiation potential, have unique advantages and great potential in producing iridoid compounds compared to other tissue cells.

[0005] There are still many problems to be solved in the existing technology for producing iridoid compounds by Rehmannia glutinosa cambium meristem stem cells. In terms of induction efficiency, the current induction methods can only make the stem cells produce limited metabolic response, and cannot fully stimulate their ability to synthesize iridoid compounds, resulting in a yield far lower than expected. Moreover, due to the lack of in-depth understanding and precise control of the complex metabolic regulation network in cells during the induction process, the yield fluctuates greatly and the stability is poor, which cannot meet the strict requirements of industrial production for product quality consistency and stability. In addition, the existing culture and induction methods usually require expensive culture medium ingredients, complex equipment and delicate operation procedures, which undoubtedly greatly increases the production cost, making the production of iridoid compounds based on stem cell technology economically unfeasible.

[0006] Therefore, developing a brand-new, efficient and economical induction method to significantly improve the ability of Rehmannia glutinosa cambium meristem stem cells to produce iridoid compounds is not only an urgent task in the field of plant biotechnology, but also a key to promoting the sustainable development and utilization of Rehmannia glutinosa medicinal resources, meeting the growing market demand and promoting the modernization development of traditional Chinese medicine. SUMMARY

[0007] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the present application proposes a method for inducing Rehmannia glutinosa stem cells to produce terpenoids. The method provided by the present application can significantly improve the yield of iridoid compounds, shorten the production cycle and reduce the production cost.

[0008] The present application provides a method for inducing Rehmannia glutinosa stem cells to produce terpenoids.

[0009] Specifically, the method for inducing Rehmannia glutinosa stem cells to produce terpenoids comprises the following steps:

[0010] (1) preparing a chitosan oligosaccharide solution; adding auxin and cytokinin in a basic MS medium to prepare a first modified MS medium; increasing the nitrogen source concentration by 5%-25%, reducing the phosphorus source concentration by 3%-15%, increasing the potassium source concentration by 3%-15%, and adding plant peptides with a concentration of 0.005-0.1 mg / L and β-cyclodextrin with a concentration of 0.5%-6% (w / v) to prepare a second modified MS medium;

[0011] (2) inoculating Rehmannia glutinosa stem cells in the first modified MS medium and culturing to the logarithmic growth phase; then soaking the logarithmic growth phase Rehmannia glutinosa stem cells in the chitosan oligosaccharide solution for 0.5-5 hours; then transferring them to the second modified MS medium and culturing under light; after the culture is completed, collecting the Rehmannia glutinosa stem cells and extracting terpenoids.

[0012] Preferably, the process of preparing the solution of chitosan oligosaccharide is: selecting chitosan oligosaccharide with a molecular weight of 800-4000 Da, dissolving in a buffer solution with a pH value of 5.5-6.5, and preparing a chitosan oligosaccharide solution with a concentration of 0.05%-0.8% (w / v). Further preferably, the process of preparing the solution of chitosan oligosaccharide is: selecting chitosan oligosaccharide with a molecular weight of 1000-3000 Da, dissolving in a buffer solution with a pH value of 5.5-6.5, and preparing a chitosan oligosaccharide solution with a concentration of 0.1%-0.6% (w / v). During the preparation process, sufficient stirring can be carried out by a magnetic stirrer to ensure uniform dispersion and dissolution. The stirring process needs to last for a certain period of time, for example, 30-60 minutes, to ensure complete dissolution of chitosan oligosaccharide, while avoiding excessive stirring to generate too much foam and affect the quality of the solution.

[0013] Preferably, in the first modified MS medium, the concentration of the auxin is 0.05-0.8 mg / L; the concentration of the cytokinin is 0.3-3 mg / L; further preferably, in the first modified MS medium, the concentration of the auxin is 0.1-0.5 mg / L; the concentration of the cytokinin is 0.5-2 mg / L.

[0014] Preferably, the auxin is naphthalene acetic acid; the cytokinin is 6-benzylaminopurine.

[0015] Preferably, in step (1), the concentration of the nitrogen source in the first modified MS medium is increased by 10%-20%, the concentration of the phosphorus source is reduced by 5%-10%, the concentration of the potassium source is increased by 5%-10%, and 0.01-0.1 mg / L of plant peptide and 1%-5% (w / v) of β-cyclodextrin are added to prepare the second modified MS medium.

[0016] Preferably, in step (2), when the dry cells of Rehmannia glutinosa are inoculated into the first modified MS medium, 1×10 4 -5×10 4 dry cells of Rehmannia glutinosa are contained in each milliliter of the first modified MS medium.

[0017] Preferably, in step (2), the culture process is: culturing for 6-15 days under the conditions of a temperature of 23-30 ℃, a light intensity of 1600-2800 lux, and a light time of 8-15 hours / day; further preferably, the culture process is: culturing for 7-14 days under the conditions of a temperature of 25-28 ℃, a light intensity of 1800 lux-2500 lux, and a light time of 10-14 hours / day.

[0018] Preferably, in step (2), the soaking time of the dry Rehmannia glutinosa stem cells in the chitosan oligosaccharide solution is 1-4 hours. The soaking process is carried out under sterile and room temperature conditions, and the culture container is gently shaken to ensure that the stem cells are in full contact with the chitosan oligosaccharide solution, so as to promote the interaction between the chitosan oligosaccharide molecules and the stem cell surface receptors, thereby effectively activating the relevant signal pathways and metabolic reactions in the cells.

[0019] Preferably, in step (2), a washing process is further included after the soaking is completed, wherein the dry Rehmannia glutinosa stem cells are washed with sterile water for 3-5 times to remove the residual chitosan oligosaccharide on the surface, so as to avoid adverse effects in the subsequent culture process.

[0020] Preferably, in step (2), the terpenoids are extracted by using an ultrasonic-assisted organic solvent extraction method. Further preferably, the process of extracting the terpenoids is as follows: ethanol is used as the solvent, the solid-liquid ratio is 1g:(8-20)mL, the ultrasonic power is 150-500W, and the ultrasonic extraction is performed for 10-40 minutes.

[0021] Preferably, in step (2), the dry Rehmannia glutinosa stem cells are collected by centrifugation, wherein the rotation speed of the centrifugation is 4000-6000rpm, and the centrifugation time is 5-10 minutes.

[0022] Compared with the prior art, the present application has the following advantages:

[0023] (1) The method provided by the present application successfully activates multiple metabolic pathways related to the synthesis of iridoid compounds in the cambium meristem stem cells of Rehmannia glutinosa through the synergistic induction of chitosan oligosaccharide, cyclodextrin and plant peptides. The culture medium is improved to better meet the metabolic needs of the stem cells in the induction stage. Compared with the traditional culture method, the method provided by the present application can increase the yield of iridoid compounds by 80%-180% and increase the purity by 11.9%-26.9%, which can significantly improve the yield of terpenoids, optimize the purity, shorten the production cycle and reduce the cost.

[0024] (2) The concentration of the inducer, the treatment process, and the formula and culture conditions of the cell culture medium are precisely optimized. On the one hand, the time required from stem cell culture to the accumulation of iridoid compounds to an observable level is shortened, and the production efficiency is significantly improved. On the other hand, by reasonably selecting and using the inducer and adjusting the proportions of nitrogen, phosphorus, potassium and other nutrients, the use amount of expensive culture medium additives is reduced while ensuring the good growth and metabolism of the stem cells, thereby reducing the production cost.

[0025] (3) The method for inducing Rehmannia glutinosa stem cells to produce terpenoids provided by the application can keep the growth and metabolism of the stem cells highly consistent and stable by precisely controlling the concentrations of chitooligosaccharides, cyclodextrins and plant peptides and optimizing the culture environment conditions. In repeated experiments, the yield fluctuation range of the iridoid terpenoids is significantly reduced, and the product quality is more stable and reliable. DETAILED DESCRIPTION

[0026] In order to make the skilled in the art more clearly understand the technical solutions described in the application, the following examples are used for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the application.

[0027] The base MS culture medium formula in the following comparative examples and examples is as follows: NH4NO3 1650 mg / L, KNO3 1900 mg / L, CaCl2·2H2O 440 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA·2H2O 37.3 mg / L, myo-inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.1 mg / L, glycine 2.0 mg / L. Other raw materials, reagents or devices can be obtained from conventional commercial channels or can be obtained by known methods, unless otherwise specified.

[0028] In the following comparative examples and examples, high performance liquid chromatography (HPLC) combined with mass spectrometry (MS) technology is used to determine the iridoid terpenoids in the extract to detect the yield and purity. The specific process is as follows:

[0029] Chromatographic column: a C18 reversed-phase chromatographic column with a particle size of 5 μm, an inner diameter of 4.6 mm and a length of 250 mm is selected.

[0030] Mobile phase: Eluent A: water (containing 0.1% formic acid), Eluent B: methanol, gradient elution, specific gradient elution conditions: methanol accounts for 20% and water accounts for 80% at the beginning, maintain for 2 minutes; then linearly increase the proportion of methanol at a rate of 5% per minute until the 20th minute when methanol accounts for 80% and water accounts for 20%; finally maintain the condition of methanol accounting for 80% and water accounting for 20% for 5 minutes to balance the chromatographic system. Flow rate: set to 1.0 mL / min; column temperature: maintain at 32°C; detection wavelength: set to 210 nm; injection volume: 20 μL.

[0031] Mass spectrometry conditions: electrospray ionization source (ESI): positive ion mode; scan range: m / z 100-1000; collision energy: optimized according to the mass-to-charge ratio (m / z) of the target compound, generally adjusted within the range of 10-50 eV to obtain the best mass spectrum signal; acquisition mode: full scan mode for obtaining the molecular ion peak and characteristic fragment ion peak of the iridoid compound for qualitative analysis; data processing: the obtained mass spectrum is analyzed by mass spectrometry software, and the target iridoid compound is qualitatively identified according to its characteristic ion peaks (such as molecular ion peak, characteristic fragment ion peak, etc.).

[0032] Comparative Example 1

[0033] Comparative Example 1 provides a traditional method for inducing Rehmannia glutinosa stem cells to produce terpenoids, which is as follows:

[0034] (1) Add 0.1 mg / L naphthaleneacetic acid and 0.5 mg / L 6-benzylaminopurine to the basic MS medium, adjust the pH value of the medium to 5.8, and sterilize under high pressure to ensure sterile conditions;

[0035] (2) Select healthy Rehmannia glutinosa stem tips as explants, and use 70% ethanol to perform surface disinfection treatment on the explants;

[0036] (3) Inoculate the explants onto the medium and culture in a constant temperature incubator with a temperature controlled at 25°C and light for 12 hours per day. Regularly check the growth and contamination of the culture.

[0037] (4) After 30 days of culture, collect the stem cells after the culture ends, and use ultrasonic-assisted organic solvent extraction method to extract iridoid compounds with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasonic power 200 W, and ultrasonic time 20 minutes. Determine the yield and purity of the iridoid compounds, and calculate the cost.

[0038] After testing, the yield of the iridoid compounds is 10 mg / g of stem cell dry weight, the purity is 67%, the cost is about 5 US dollars per gram of stem cell dry weight (including medium, hormone, energy and labor cost), and the production cycle is 30 days.

[0039] Example 1

[0040] The method for inducing Rehmannia glutinosa stem cells to produce terpenoids comprises the following steps:

[0041] (1) Chitosan solution preparation: chitosan with a molecular weight of 1000 Da is selected and dissolved in a sterilized phosphate buffer with a pH of 5.8 to prepare a chitosan solution with a concentration of 0.1% (w / v);

[0042] Preparation of the first modified MS medium: auxin and cytokinin are added to the basic MS medium to make it contain 0.1 mg / L naphthalene acetic acid and 0.5 mg / L 6-benzylaminopurine;

[0043] Preparation of the second modified MS medium: the concentration of nitrogen source in the first modified MS medium is increased by 10%, the concentration of phosphorus source is decreased by 5%, the concentration of potassium source is increased by 5%, and 0.01 mg / L plant peptide and 1% (w / v) β-cyclodextrin are added;

[0044] (2) The cambium meristem stem cells of Rehmannia glutinosa are inoculated in the first modified MS medium and cultured to the logarithmic growth phase;

[0045] (3) The logarithmic growth phase stem cells are soaked in the chitosan solution for 1 hour by the soaking method, and then washed with sterile water. Then they are transferred to the second modified MS medium and cultured for 7 days under the conditions of a temperature of 25°C, a light intensity of 1800 lux, and a light time of 10 hours per day. During this period, the growth state of the stem cells is observed regularly, and the cell morphology and proliferation are checked by a microscope to ensure their healthy growth. If pollution or abnormal growth is found, appropriate measures are taken in time.

[0046] (4) Product extraction: after the culture is completed, the stem cells are collected, and the ultrasonic-assisted organic solvent extraction method is used to extract the iridoid compounds with ethanol as the solvent (the solid-liquid ratio is 1 g:10 mL), the ultrasonic power is 200 W, and the ultrasonic time is 20 minutes.

[0047] It is detected that the yield of the iridoid compounds reaches 15 mg / g of the dry weight of the stem cells, and the purity is 75%. Compared with the traditional culture method in Comparative Example 1, the yield is increased by 50%, the purity is increased by 11.9%, the production cycle is shortened by about 23 days, and the cost is reduced by 15%.

[0048] Example 2

[0049] The method for inducing Rehmannia glutinosa stem cells to produce terpenoids comprises the following steps:

[0050] (1) Chitosan oligosaccharide solution preparation: Chitosan oligosaccharide with a molecular weight of 2000 Da was selected and dissolved in a sterilized phosphate buffer with a pH of 6.0 to prepare a chitosan oligosaccharide solution with a concentration of 0.3% (w / v);

[0051] Preparation of the first modified MS medium: auxin and cytokinin were added to the basic MS medium to contain 0.3 mg / L naphthalene acetic acid and 1.0 mg / L 6-benzylaminopurine;

[0052] Preparation of the second modified MS medium: the nitrogen source concentration in the first modified MS medium was increased by 15%, the phosphorus source concentration was reduced by 8%, the potassium source concentration was increased by 8%, and 0.05 mg / L plant peptide and 3% (w / v) β-cyclodextrin were added;

[0053] (2) The cambium meristem stem cells of Rehmannia glutinosa were inoculated in the first modified MS medium and cultured to the logarithmic growth phase;

[0054] (3) The logarithmic growth phase stem cells were soaked in the chitosan oligosaccharide solution for 2 hours by the soaking method, and then washed with sterile water. Then they were transferred to the second modified MS medium and cultured for 10 days under the conditions of a temperature of 26°C, a light intensity of 2000 lux, and a light time of 12 hours / day. During this period, the growth state of the stem cells was observed regularly, and the cell morphology and proliferation were checked by microscope to ensure their healthy growth. If pollution or abnormal growth was found, appropriate measures were taken in time.

[0055] (4) Product extraction: after the culture ended, the stem cells were collected, and the ultrasonic-assisted organic solvent extraction method was used to extract the iridoid compounds with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasonic power 200 W, and ultrasonic time 20 minutes.

[0056] The yield of the iridoid compounds reached 22 mg / g dry weight of stem cells, and the purity was 80%. Compared with the traditional culture method in Comparative Example 1, the yield was increased by 120%, the purity was increased by 19.4%, the production cycle was shortened by about 20 days, and the cost was reduced by 20%.

[0057] Example 3

[0058] The method for inducing Rehmannia glutinosa stem cells to produce terpenoids comprises the following steps:

[0059] (1) Chitosan oligosaccharide solution preparation: Chitosan oligosaccharide with a molecular weight of 3000 Da was selected and dissolved in a sterilized phosphate buffer with a pH of 6.2 to prepare a chitosan oligosaccharide solution with a concentration of 0.5% (w / v);

[0060] Preparation of the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.5 mg / L naphthalene acetic acid and 2.0 mg / L 6-benzylaminopurine;

[0061] Preparation of the second modified MS medium: increase the nitrogen source concentration by 20%, reduce the phosphorus source concentration by 10%, increase the potassium source concentration by 10%, and add plant peptides with a concentration of 0.1 mg / L and β-cyclodextrin with a concentration of 5% (w / v) to the first modified MS medium;

[0062] (2) Inoculate the phellem cambium meristem stem cells into the first modified MS medium and culture to the logarithmic growth phase;

[0063] (3) Soak the logarithmic growth phase stem cells in a chitosan solution for 3 hours using the soaking method, then wash them with sterile water after taking them out; then transfer them to the second modified MS medium and culture for 14 days under the conditions of a temperature of 28°C, a light intensity of 2500 lux, and a light time of 14 hours / day. During this period, observe the growth state of the stem cells regularly, check the cell morphology and proliferation by microscope, and ensure their healthy growth. If pollution or abnormal growth is found, take appropriate measures in time.

[0064] (4) Product extraction: after the culture ends, collect the stem cells and use the ultrasonic-assisted organic solvent extraction method to extract iridoid compounds with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasonic power 200 W, and ultrasonic time 20 minutes.

[0065] After detection, the yield of iridoid compounds reaches 25 mg / g dry weight of stem cells, and the purity is 82%. Compared with the traditional culture method in Comparative Example 1, the yield is increased by 150%, the purity is increased by 22.4%, the production cycle is shortened by about 16 days, and the cost is reduced by 22%.

[0066] Example 4

[0067] The method for inducing Rehmannia glutinosa stem cells to produce terpenoids includes the following steps:

[0068] (1) Preparation of chitosan solution: select chitosan with a molecular weight of 1500 Da, dissolve it in a sterilized phosphate buffer with a pH value of 5.9, and prepare a chitosan solution with a concentration of 0.2% (w / v);

[0069] Preparation of the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.2 mg / L naphthalene acetic acid and 0.8 mg / L 6-benzylaminopurine;

[0070] Preparation of the second modified MS medium: in the first modified MS medium, the concentration of nitrogen source is increased by 12%, the concentration of phosphorus source is decreased by 6%, the concentration of potassium source is increased by 6%, and 0.03 mg / L of plant peptide and 2% (w / v) of β-cyclodextrin are added;

[0071] (2) The stem cell of the root of rehmannia meristem is inoculated in the first modified MS medium and cultured to the logarithmic growth phase;

[0072] (3) The stem cell in the logarithmic growth phase is soaked in the chitosan oligosaccharide solution for 1.5 hours by the soaking method, and then washed with sterile water. Then, it is transferred to the second modified MS medium and cultured for 8 days under the conditions of a temperature of 25.5°C, a light intensity of 1900 lux, and a light time of 11 hours per day. During the period, the growth state of the stem cell is observed regularly, the cell morphology and proliferation are checked by a microscope to ensure the healthy growth, and corresponding treatment measures are taken in time if pollution or abnormal growth is found.

[0073] (4) Product extraction: after the culture, the stem cell is collected, and the iridoid compound is extracted by the ultrasonic-assisted organic solvent extraction method with ethanol as the solvent (the solid-liquid ratio is 1 g:10 mL), an ultrasonic power of 200 W, and an ultrasonic time of 20 minutes.

[0074] Through detection, the yield of the iridoid compound reaches 18 mg / g of the dry weight of the stem cell, and the purity is 78%. Compared with the traditional culture method in Comparative Example 1, the yield is increased by 80%, the purity is increased by 16.4%, the production cycle is shortened by about 22 days, and the cost is reduced by 18%.

[0075] Example 5

[0076] The method for inducing the stem cell of the root of rehmannia to produce the terpenoid compound comprises the following steps:

[0077] (1) Preparation of the chitosan oligosaccharide solution: chitosan oligosaccharide with a molecular weight of 2500 Da is selected, dissolved in a sterilized phosphate buffer with a pH value of 6.1, and prepared into a chitosan oligosaccharide solution with a concentration of 0.4% (w / v);

[0078] Preparation of the first modified MS medium: in the basic MS medium, auxin and cytokinin are added so that it contains 0.4 mg / L of naphthalene acetic acid and 1.5 mg / L of 6-benzylaminopurine;

[0079] Preparation of the second modified MS medium: in the first modified MS medium, the concentration of nitrogen source is increased by 18%, the concentration of phosphorus source is decreased by 9%, the concentration of potassium source is increased by 9%, and 0.07 mg / L of plant peptide and 4% (w / v) of β-cyclodextrin are added;

[0080] (2) Replant the stem cells in the logarithmic growth phase into the first modified MS medium, and culture until the logarithmic growth phase;

[0081] (3) Soak the stem cells in the logarithmic growth phase in a chitosan solution for 2.5 hours, and then rinse them with sterile water. Then, transfer the stem cells to the second modified MS medium, and culture for 12 days under the conditions of a temperature of 27°C, a light intensity of 2200 lux, and a light time of 13 hours per day. During the culture, observe the growth state of the stem cells regularly, and check the cell morphology and proliferation by microscopy to ensure the healthy growth of the stem cells. If pollution or abnormal growth is found, take appropriate measures in time.

[0082] (4) Product extraction: After the culture, collect the stem cells, and extract the iridoid compounds by the ultrasonic-assisted organic solvent extraction method using ethanol as the solvent (the solid-liquid ratio is 1 g:10 mL), an ultrasonic power of 200 W, and an ultrasonic time of 20 minutes.

[0083] The yield of the iridoid compounds reaches 20 mg / g of the dry weight of the stem cells, and the purity is 81%. Compared with the traditional culture method in Comparative Example 1, the yield is increased by 100%, the purity is increased by 20.9%, the production cycle is shortened by about 18 days, and the cost is reduced by 20%.

[0084] Example 6

[0085] The method for inducing the stem cells of Rehmannia glutinosa to produce terpenoids includes the following steps:

[0086] (1) Preparation of a chitosan solution: select chitosan with a molecular weight of 2000 Da, dissolve it in a sterilized phosphate buffer with a pH value of 6.0, and prepare a chitosan solution with a concentration of 0.4% (w / v);

[0087] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium so that it contains 0.3 mg / L naphthalene acetic acid and 1.2 mg / L 6-benzylaminopurine;

[0088] Prepare the second modified MS medium: increase the nitrogen source concentration by 16%, decrease the phosphorus source concentration by 7%, increase the potassium source concentration by 7%, and add plant peptides with a concentration of 0.06 mg / L and β-cyclodextrin with a concentration of 3% (w / v) to the first modified MS medium;

[0089] (2) Replant the stem cells in the logarithmic growth phase into the first modified MS medium, and culture until the logarithmic growth phase;

[0090] (3) Adopting soaking method to put the stem cells in logarithmic growth phase into chitosan solution for 3 hours, then wash them with sterile water after taking out; then transfer them to the second modified MS medium, and cultivate them for 11 days under the conditions of temperature 26.5℃, light intensity 2100 lux, and light time 12.5 hours per day. During this period, observe the growth state of the stem cells regularly, check the cell morphology and proliferation by microscope, and ensure their healthy growth. If pollution or abnormal growth is found, take corresponding measures in time.

[0091] (4) Product extraction: after the cultivation, collect the stem cells, and adopt ultrasonic-assisted organic solvent extraction method to extract iridoid compounds with ethanol as solvent (material-solvent ratio 1g:10mL), ultrasonic power 200W, and ultrasonic time 20 minutes.

[0092] Through detection, the yield of iridoid compounds reaches 28mg / g stem cell dry weight, and the purity is 85%. Compared with the traditional cultivation method in Comparative Example 1, the yield is increased by 180%, the purity is increased by 26.9%, the production cycle is shortened by about 19 days, and the cost is reduced by 25%.

[0093] Comparative Example 2

[0094] The method for inducing Rehmannia glutinosa stem cells to produce terpenoids comprises the following steps:

[0095] (1) Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium, so that it contains 0.3mg / L naphthalene acetic acid and 1.2mg / L 6-benzylaminopurine;

[0096] Prepare the second modified MS medium: increase the nitrogen source concentration by 16%, decrease the phosphorus source concentration by 7%, increase the potassium source concentration by 7% in the first modified MS medium, and add plant peptides with a concentration of 0.06mg / L and β-cyclodextrin with a concentration of 3%(w / v);

[0097] (2) Inoculate the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium, and cultivate them to the logarithmic growth phase;

[0098] (3) Transfer the stem cells in logarithmic growth phase to the second modified MS medium, and cultivate them for 11 days under the conditions of temperature 26.5℃, light intensity 2100 lux, and light time 12.5 hours per day. During this period, observe the growth state of the stem cells regularly, check the cell morphology and proliferation by microscope, and ensure their healthy growth. If pollution or abnormal growth is found, take corresponding measures in time.

[0099] (4) Product extraction: after the end of the culture, the stem cells were collected, and the ultrasonic-assisted organic solvent extraction method was used to extract the iridoid compounds with ethanol as the solvent (solid-liquid ratio 1 g: 10 mL), ultrasonic power 200 W, and ultrasonic time 20 minutes.

[0100] It was detected that the yield of the iridoid compounds reached 12 mg / g dry weight of stem cells, and the purity was 72%.

[0101] Comparative Example 3

[0102] The method for inducing the stem cells of Rehmannia glutinosa to produce terpenoids comprises the following steps:

[0103] (1) Preparation of chitosan solution: chitosan with a molecular weight of 2000 Da was selected, dissolved in a sterilized phosphate buffer with a pH value of 6.0, and prepared into a chitosan solution with a concentration of 0.4% (w / v);

[0104] Preparation of the first modified MS culture medium: auxin and cytokinin were added to the basic MS culture medium, so that it contained 0.3 mg / L naphthalene acetic acid and 1.2 mg / L 6-benzylaminopurine;

[0105] Preparation of the second modified MS culture medium: the nitrogen source concentration in the first modified MS culture medium was increased by 16%, the phosphorus source concentration was decreased by 7%, the potassium source concentration was increased by 7%, and β-cyclodextrin with a concentration of 3% (w / v) was added;

[0106] (2) The stem cells of the cambium meristem of Rehmannia glutinosa were inoculated in the first modified MS culture medium and cultured to the logarithmic growth phase;

[0107] (3) The stem cells in the logarithmic growth phase were soaked in the chitosan solution for 3 hours by using the soaking method, and then washed with sterile water after being taken out; then they were transferred to the second modified MS culture medium and cultured for 11 days under the conditions of a temperature of 26.5°C, a light intensity of 2100 lux, and a light time of 12.5 hours / day. During this period, the growth state of the stem cells was observed regularly, and the cell morphology and proliferation were checked by a microscope to ensure their healthy growth. If pollution or abnormal growth was found, appropriate treatment measures were taken in time.

[0108] (4) Product extraction: after the end of the culture, the stem cells were collected, and the ultrasonic-assisted organic solvent extraction method was used to extract the iridoid compounds with ethanol as the solvent (solid-liquid ratio 1 g: 10 mL), ultrasonic power 200 W, and ultrasonic time 20 minutes.

[0109] It was detected that the yield of the iridoid compounds reached 12.5 mg / g dry weight of stem cells, and the purity was 69%.

[0110] Comparative Example 4

[0111] A method for inducing Rehmannia glutinosa stem cells to produce terpenoids, comprising the following steps:

[0112] (1) Chitosan solution preparation: chitosan with a molecular weight of 2000 Da is selected and dissolved in a sterilized phosphate buffer with a pH of 6.0 to prepare a chitosan solution with a concentration of 0.4% (w / v);

[0113] Preparation of the first modified MS medium: auxin and cytokinin are added to the basic MS medium to make it contain 0.3 mg / L naphthalene acetic acid and 1.2 mg / L 6-benzylaminopurine;

[0114] Preparation of the second modified MS medium: the concentration of nitrogen source in the first modified MS medium is increased by 16%, the concentration of phosphorus source is decreased by 7%, the concentration of potassium source is increased by 7%, and plant peptide with a concentration of 0.06 mg / L is added;

[0115] (2) The cambium meristem stem cells of Rehmannia glutinosa are inoculated in the first modified MS medium and cultured to the logarithmic growth phase;

[0116] (3) The logarithmic growth phase stem cells are soaked in the chitosan solution for 3 hours by using the soaking method, and then washed with sterile water after being taken out; and then transferred to the second modified MS medium, and cultured for 11 days under the conditions of a temperature of 26.5℃, a light intensity of 2100 lux, and a light time of 12.5 hours / day. During this period, the growth state of the stem cells is observed regularly, the cell morphology and proliferation are checked by a microscope to ensure the healthy growth, and corresponding treatment measures are taken in time if pollution or abnormal growth is found.

[0117] (4) Product extraction: after the culture is completed, the stem cells are collected, and the ultrasonic-assisted organic solvent extraction method is used to extract the iridoid compounds with ethanol as the solvent (the solid-liquid ratio is 1 g:10 mL), the ultrasonic power is 200 W, and the ultrasonic time is 20 minutes.

[0118] After detection, the yield of the iridoid compounds reaches 13.5 mg / g of stem cell dry weight, and the purity is 73%.

[0119] Comparative Example 5

[0120] A method for inducing Rehmannia glutinosa stem cells to produce terpenoids, comprising the following steps:

[0121] (1) Chitosan solution preparation: chitosan with a molecular weight of 2000 Da is selected and dissolved in a sterilized phosphate buffer with a pH of 6.0 to prepare a chitosan solution with a concentration of 0.4% (w / v);

[0122] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium to contain 0.3 mg / L naphthalene acetic acid and 1.2 mg / L 6-benzylaminopurine;

[0123] Prepare the second modified MS medium: add plant peptide at a concentration of 0.06 mg / L and β-cyclodextrin at a concentration of 3% (w / v) to the first modified MS medium;

[0124] (2) inoculate the phloem cambium meristem stem cells into the first modified MS medium and culture to the logarithmic growth phase;

[0125] (3) immerse the logarithmic growth phase stem cells in a chitosan solution for 3 hours by the immersion method, then wash with sterile water after taking out; then transfer them to the second modified MS medium and culture for 11 days under the conditions of a temperature of 26.5℃, a light intensity of 2100 lux, and a light time of 12.5 hours per day. During this period, observe the growth state of the stem cells regularly, check the cell morphology and proliferation by microscope, and ensure their healthy growth. If pollution or abnormal growth is found, take corresponding measures in time.

[0126] (4) product extraction: after the culture, collect the stem cells and extract iridoid compounds by the ultrasonic-assisted organic solvent extraction method with ethanol as the solvent (the solid-liquid ratio is 1 g:10 mL), an ultrasonic power of 200 W, and an ultrasonic time of 20 minutes.

[0127] After detection, the yield of iridoid compounds reaches 18 mg / g stem cell dry weight, and the purity is 81%.

[0128] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as the limitation of the patent scope of the present application. It should be noted that for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims.

Claims

1. A method for inducing Rehmannia glutinosa stem cells to produce terpenoids, characterized in that, The method comprises the following steps: (1) preparing a chitosan oligosaccharide solution with a concentration of 0.05%-0.8%(w / v); adding auxin and cytokinin into a basic MS culture medium to prepare a first modified MS culture medium; increasing the concentration of nitrogen source in the first modified MS culture medium by 12%-25%, decreasing the concentration of phosphorus source by 6%-15%, increasing the concentration of potassium source by 6%-15%, and adding plant peptide with a concentration of 0.03-0.1mg / L and β-cyclodextrin with a concentration of 2%-6%(w / v) to prepare a second modified MS culture medium; (2) inoculating dry rehmannia cells into the first modified MS culture medium and culturing until the logarithmic growth phase; then immersing the logarithmic growth phase dry rehmannia cells in the chitosan oligosaccharide solution for 0.5-5 hours; then transferring the dry rehmannia cells into the second modified MS culture medium and culturing under illumination; after the culture, collecting the dry rehmannia cells and extracting terpenoids.

2. The method of claim 1, wherein, The process of preparing the chitosan oligosaccharide solution is as follows: selecting chitosan oligosaccharide with a molecular weight of 800-4000Da and dissolving it in a buffer with a pH value of 5.5-6.

5.

3. The method according to claim 1 or 2, characterized in that, In the first modified MS culture medium, the concentration of the auxin is 0.05-0.8mg / L; and the concentration of the cytokinin is 0.3-3mg / L.

4. The method of claim 3, wherein, In the first modified MS culture medium, the concentration of the auxin is 0.1-0.5mg / L; and the concentration of the cytokinin is 0.5-2mg / L.

5. The method of claim 4, wherein, The auxin is naphthalene acetic acid; and the cytokinin is 6-benzylaminopurine.

6. The method of claim 1 or 2, wherein, In step (1), the concentration of the nitrogen source in the first modified MS culture medium is increased by 12%-20%, the concentration of the phosphorus source is decreased by 6%-10%, the concentration of the potassium source is increased by 6%-10%, and plant peptide with a concentration of 0.03-0.1mg / L and β-cyclodextrin with a concentration of 2%-5%(w / v) are added to prepare the second modified MS culture medium.

7. The method according to claim 1 or 2, characterized in that, In step (2), the drynaria stem cells are inoculated into the first modified MS medium at a concentration of 1 x 10 4 -5 x 10 4 cells per milliliter of the first modified MS medium.

8. The method of claim 1 or 2, wherein, In step (2), the culture is carried out at a temperature of 23-30℃, an illumination intensity of 1600-2800lux, and an illumination time of 8-15 hours / day for 6-15 days.

9. The method of claim 1 or 2, wherein, In step (2), the immersion time of the dry rehmannia cells in the chitosan oligosaccharide solution is 1-4 hours.

10. The method of claim 1, wherein, In step (2), the terpenoids are extracted by ultrasonic-assisted organic solvent extraction.