Method for inducing rehmannia stem cells to produce terpenoids

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses a method for inducing rehmannia stem cells to produce terpenoids. The method comprises the following steps: preparing a chitosan oligosaccharide solution; auxin and cytokinin are added into the basic MS culture medium, and a first improved MS culture medium is prepared; adjusting the concentrations of a nitrogen source, a phosphorus source and a potassium source in the first improved MS culture medium, and adding plant peptide and beta-cyclodextrin to prepare a second improved MS culture medium; inoculating the rehmannia stem cells into a first improved MS culture medium, and culturing to a logarithmic phase; then soaking the bamboo shoots in a chitosan oligosaccharide solution for 0.5-5 hours; transferring the culture medium into a second improved MS culture medium, and culturing under illumination; and after the culture is finished, collecting the rehmannia stem cells, and extracting the terpenoids. The method provided by the invention can obviously improve the yield of the terpenoids, optimize the purity, shorten the production cycle and reduce the cost.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds. Background Art

[0002] Rehmannia root occupies a pivotal position in the history of traditional Chinese medicine, and its medicinal value has long been widely recognized and applied. Among them, iridoid compounds are the core components of Rehmannia root to exert many pharmacological effects, showing significant activity in anti-oxidation, anti-inflammatory, neuroprotection and immune regulation, and playing an important role in treating various diseases and maintaining human health.

[0003] For a long time, the industrial acquisition of cyclopentadiene 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 affected by various complex environmental factors during its natural growth. Different soil textures, fertility levels, pH, and variable climatic conditions, including temperature, humidity, light duration and intensity, will lead to significant differences in the growth conditions of Rehmannia glutinosa plants and the accumulation of secondary metabolites. This makes the content of cyclopentadiene compounds extremely unstable, and it is difficult to ensure a stable supply of production. 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 production costs and time costs, and seriously limits its large-scale and efficient industrial production.

[0004] Entering the era of modern biotechnology, the rise of plant tissue culture technology has brought hope for solving this dilemma. In particular, the emergence of plant stem cell technology has made it possible to produce secondary metabolites of medicinal plants in a controlled environment in vitro. Compared with other tissue cells, stem cells of the meristem tissue of the cambium of Rehmannia glutinosa have unique advantages and great potential in the production of cyclopentadiene ether terpenoids due to their high self-renewal ability and multidirectional differentiation potential.

[0005] There are still many problems to be solved in the existing technical methods for producing cyclopentadiene compounds from Rehmannia glutinosa cambium meristem stem cells. In terms of induction efficiency, the induction methods currently used can often only cause stem cells to produce limited metabolic responses and cannot fully stimulate their ability to synthesize cyclopentadiene compounds, resulting in production far below expected levels. Moreover, due to the lack of in-depth understanding and precise control of the complex metabolic regulatory network within the cell during the induction process, the output in the production process fluctuates greatly and the stability is poor, making it difficult to meet the strict requirements of industrial production for product quality consistency and stability. In addition, existing culture and induction methods usually require expensive culture medium components, complex equipment, and sophisticated operating procedures, which undoubtedly greatly increases production costs, making the economic feasibility of cyclopentadiene compound production based on stem cell technology greatly reduced.

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

[0007] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds. The method provided by the present invention can significantly increase the yield of terpenoid compounds, shorten the production cycle, and reduce production costs.

[0008] The invention provides a method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds.

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

[0010] (1) preparing a chitosan oligosaccharide solution; adding auxin and cytokinin to a basic MS medium to prepare a first improved MS medium; increasing the nitrogen source concentration in the first improved MS medium by 5%-25%, reducing the phosphorus source concentration by 3%-15%, increasing the potassium source concentration by 3%-15%, and adding a plant peptide 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 improved MS medium;

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

[0012] Preferably, the process of preparing the chitosan oligosaccharide solution is: selecting chitosan oligosaccharide with a molecular weight of 800-4000Da, dissolving it 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 chitosan oligosaccharide solution is: selecting chitosan oligosaccharide with a molecular weight of 1000-3000Da, dissolving it 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, a magnetic stirrer can be used to fully stir it so that it is evenly dispersed and dissolved. The stirring process needs to last for a certain time, such as 30-60 minutes, to ensure that the chitosan oligosaccharide is completely dissolved, while avoiding excessive stirring to produce too much foam that affects the quality of the solution.

[0013] Preferably, in the first modified MS culture 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 culture 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 naphthylacetic acid; and the cytokinin is 6-benzylaminopurine.

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

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

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

[0018] Preferably, in step (2), the soaking time of the Rehmannia stem cells in the chitosan oligosaccharide solution is 1-4 hours. The soaking process needs to be carried out under sterile and room temperature conditions, and the culture container is gently shaken to allow the stem cells to fully contact with the chitosan oligosaccharide solution, promote the interaction between the chitosan oligosaccharide molecules and the stem cell surface receptors, and effectively activate 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, and the washing process is: the Rehmannia stem cells are rinsed with sterile water for 3-5 times to remove residual chitosan oligosaccharides on the surface to avoid adverse effects thereof in subsequent culture processes.

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

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

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The method provided by the present invention successfully activates multiple metabolic pathways related to the synthesis of iridoid compounds in the stem cells of the meristem of the cambium of Rehmannia glutinosa through the synergistic induction of oligochitosan, cyclodextrin and plant peptides; and improves the culture medium to make it more in line with the metabolic needs of stem cells in the induction stage. Compared with the traditional culture method, the method provided by the present invention can increase the yield of iridoid compounds by 80%-180% and the purity by 11.9%-26.9%, which can significantly increase the yield of terpenoid compounds, optimize the purity, shorten the production cycle and reduce costs.

[0024] (2) The present invention has precisely optimized the concentration of the inducing substance, the treatment process, and the medium formula and culture conditions for cell culture. On the one hand, the time required from stem cell culture to the accumulation of cyclopentadiene ether terpenoid compounds to a considerable level is shortened, significantly improving production efficiency. On the other hand, by rationally selecting and using inducing substances and adjusting the ratio of nutrients such as nitrogen, phosphorus, and potassium, while ensuring good growth and metabolism of stem cells, the use of expensive culture medium additives is reduced, thereby reducing production costs.

[0025] (3) The method of inducing Rehmannia stem cells to produce terpenoids provided by the present invention can maintain a high degree of consistency and stability in the growth and metabolic processes of stem cells by precisely controlling the concentrations of chitosan oligosaccharides, cyclodextrins and plant peptides, and optimizing the culture environment conditions. In repeated experiments, the fluctuation range of the production of terpenoids was significantly reduced, and the product quality was more stable and reliable. DETAILED DESCRIPTION

[0026] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0027] The basic MS medium formula in the following comparative examples and embodiments is as follows: NH 4 NO 3 1650mg / L, KNO 3 1900mg / L, CaCl 2 ·2H 2 O 440mg / L, MgSO 4 7H 2 O 370mg / L, KH 2 PO 4 170mg / L; KI 0.83mg / L, H 3 BO 3 6.2mg / L, MnSO4·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O 8.6mg / L, Na 2 MoO 4 ·2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O0.025mg / L, CoCl 2 6H 2 O 0.025mg / L, FeSO 4 7H 2 O 27.8mg / L, Na 2 -EDTA·2H 2 O 37.3 mg / L, inositol 100 mg / L, niacin 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.1 mg / L, glycine 2.0 mg / L. Unless otherwise specified, other raw materials, reagents or equipment can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0028] In the following comparative examples and embodiments, the iridoid compounds in the extracts were determined by high performance liquid chromatography (HPLC) combined with mass spectrometry (MS) to detect their yield and purity. The details are as follows:

[0029] Chromatographic column: C18 reverse phase column with particle size of 5 μm, inner diameter of 4.6 mm and length of 250 mm was selected.

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

[0031] Mass spectrometry conditions: electrospray ionization source (ESI): positive ion mode; scanning 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 spectrometry signal; acquisition mode: full scan mode, used to obtain the molecular ion peaks and characteristic fragment ion peaks of cyclopentadiene compounds for qualitative analysis; data processing: the obtained mass spectrum is analyzed by mass spectrometry software, and qualitative identification is performed based on the characteristic ion peaks of the target cyclopentadiene compounds (such as molecular ion peaks, characteristic fragment ion peaks, etc.).

[0032] Comparative Example 1

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

[0034] (1) 0.1 mg / L naphthaleneacetic acid and 0.5 mg / L 6-benzylaminopurine were added to the basic MS medium, the pH value of the medium was adjusted to 5.8, and high pressure sterilization was performed to ensure sterile conditions;

[0035] (2) Select healthy stem tips of Rehmannia glutinosa as explants and use 70% ethanol to disinfect the surface of the explants;

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

[0037] (4) After 30 days of culture, stem cells were collected and cyclopentadienyl compounds were extracted by ultrasound-assisted organic solvent extraction, using ethanol as solvent (solid-liquid ratio 1 g:10 mL), ultrasound power 200 W, and ultrasound time 20 minutes. The yield and purity of cyclopentadienyl compounds were determined, and the cost was calculated.

[0038] After testing, the yield of cyclopentadiene ether terpenoid compounds is 10 mg / g dry cell dry weight, with a purity of 67%. The cost is about US$5 / gram dry cell dry weight (including culture medium, hormones, energy and labor costs), and the production cycle is 30 days.

[0039] Example 1

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

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

[0042] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.1 mg / L naphthaleneacetic acid and 0.5 mg / L 6-benzylaminopurine;

[0043] The second modified MS medium was prepared by increasing the nitrogen source concentration by 10%, decreasing the phosphorus source concentration by 5%, increasing the potassium source concentration by 5%, and adding 0.01 mg / L of plant peptide and 1% (w / v) of β-cyclodextrin to the first modified MS medium;

[0044] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0045] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 1 hour by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 7 days at a temperature of 25°C, a light intensity of 1800 lux, and a light time of 10 hours / day. During this period, the growth status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0046] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0047] After testing, the yield of cyclopentadiene ether terpenoids reached 15 mg / g dry cell weight, and the purity was 75%. Compared with the traditional culture method in comparative example 1, the yield was increased by 50%, the purity was increased by 11.9%, the production cycle was shortened by about 23 days, and the cost was reduced by 15%.

[0048] Example 2

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

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

[0051] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.3 mg / L naphthaleneacetic acid and 1.0 mg / L 6-benzylaminopurine;

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

[0053] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0054] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 2 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 10 days at a temperature of 26°C, a light intensity of 2000 lux, and a light time of 12 hours / day. During this period, the growth status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0055] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0056] After testing, the yield of cyclopentadiene ether terpenoids reached 22 mg / g dry cell weight, and the purity was 80%. Compared with the traditional culture method in comparative example 1, the yield increased by 120%, the purity 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 terpenoid compounds comprises the following steps:

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

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

[0061] The second modified MS medium was prepared by increasing the nitrogen source concentration by 20%, decreasing the phosphorus source concentration by 10%, increasing the potassium source concentration by 10%, and adding 0.1 mg / L of plant peptide and 5% (w / v) of β-cyclodextrin to the first modified MS medium;

[0062] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0063] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 3 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 14 days at a temperature of 28°C, a light intensity of 2500 lux, and a light time of 14 hours / day. During this period, the growth status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0064] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0065] After testing, the yield of cyclopentadiene ether terpenoids reached 25 mg / g dry cell weight, and the purity was 82%. Compared with the traditional culture method in comparative example 1, the yield increased by 150%, the purity increased by 22.4%, the production cycle was shortened by about 16 days, and the cost was reduced by 22%.

[0066] Example 4

[0067] The method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds comprises the following steps:

[0068] (1) Preparation of chitosan oligosaccharide solution: chitosan oligosaccharide with a molecular weight of 1500 Da was selected and dissolved in a sterilized phosphate buffer with a pH value of 5.9 to prepare a chitosan oligosaccharide solution with a concentration of 0.2% (w / v);

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

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

[0071] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0072] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 1.5 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 8 days at a temperature of 25.5°C, a light intensity of 1900 lux, and a light time of 11 hours / day. During this period, the growth status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0073] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0074] After testing, the yield of cyclopentadiene ether terpenoids reached 18 mg / g dry cell weight, and the purity was 78%. Compared with the traditional culture method in comparative example 1, the yield increased by 80%, the purity increased by 16.4%, the production cycle was shortened by about 22 days, and the cost was reduced by 18%.

[0075] Example 5

[0076] The method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds comprises the following steps:

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

[0078] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.4 mg / L naphthaleneacetic acid and 1.5 mg / L 6-benzylaminopurine;

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

[0080] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0081] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 2.5 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 12 days at a temperature of 27°C, a light intensity of 2200 lux, and a light time of 13 hours / day. During this period, the growth status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0082] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0083] After testing, the yield of cyclopentadiene ether terpenoids reached 20 mg / g dry cell weight, and the purity was 81%. Compared with the traditional culture method in comparative example 1, the yield increased by 100%, the purity increased by 20.9%, the production cycle was shortened by about 18 days, and the cost was reduced by 20%.

[0084] Example 6

[0085] The method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds comprises the following steps:

[0086] (1) Preparation of chitosan oligosaccharide solution: chitosan oligosaccharide with a molecular weight of 2000 Da was selected and dissolved in a sterilized phosphate buffer with a pH value of 6.0 to prepare a chitosan oligosaccharide 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 to make it contain 0.3 mg / L naphthaleneacetic acid and 1.2 mg / L 6-benzylaminopurine;

[0088] The second modified MS medium was prepared: the nitrogen source concentration in the first modified MS medium was increased by 16%, the phosphorus source concentration was decreased by 7%, the potassium source concentration was increased by 7%, and a plant peptide with a concentration of 0.06 mg / L and β-cyclodextrin with a concentration of 3% (w / v) were added;

[0089] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0090] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 3 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 11 days at 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 status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0091] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0092] After testing, the yield of cyclopentadiene ether terpenoids reached 28 mg / g dry cell weight, and the purity was 85%. Compared with the traditional culture method in comparative example 1, the yield increased by 180%, the purity increased by 26.9%, the production cycle was shortened by about 19 days, and the cost was reduced by 25%.

[0093] Comparative Example 2

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

[0095] (1) preparing a first modified MS medium: adding auxin and cytokinin to the basic MS medium to make it contain 0.3 mg / L naphthaleneacetic acid and 1.2 mg / L 6-benzylaminopurine;

[0096] The second modified MS medium was prepared: the nitrogen source concentration in the first modified MS medium was increased by 16%, the phosphorus source concentration was decreased by 7%, the potassium source concentration was increased by 7%, and a plant peptide with a concentration of 0.06 mg / L and β-cyclodextrin with a concentration of 3% (w / v) were added;

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

[0098] (3) Transfer the stem cells in the logarithmic growth phase to the second modified MS medium and culture them for 11 days at a temperature of 26.5°C, a light intensity of 2100 lux, and a light exposure time of 12.5 hours / day. During this period, regularly observe the growth status of the stem cells, and examine the cell morphology and proliferation under a microscope to ensure their healthy growth. If contamination or abnormal growth is found, take appropriate treatment measures in a timely manner.

[0099] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0100] After testing, the yield of cyclopentadiene ether terpenoid compounds reached 12 mg / g dry cell dry weight, and the purity was 72%.

[0101] Comparative Example 3

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

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

[0104] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.3 mg / L naphthaleneacetic acid and 1.2 mg / L 6-benzylaminopurine;

[0105] The second modified MS medium was prepared by increasing the nitrogen source concentration by 16%, decreasing the phosphorus source concentration by 7%, increasing the potassium source concentration by 7%, and adding 3% (w / v) β-cyclodextrin in the first modified MS medium;

[0106] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0107] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 3 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 11 days at 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 status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0108] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0109] After testing, the yield of cyclopentadiene ether terpenoid compounds reached 12.5 mg / g dry cell dry weight, and the purity was 69%.

[0110] Comparative Example 4

[0111] The method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds comprises the following steps:

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

[0113] Prepare the first modified MS medium: add auxin and cytokinin to the basic MS medium to make it contain 0.3 mg / L naphthaleneacetic acid and 1.2 mg / L 6-benzylaminopurine;

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

[0115] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0116] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 3 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 11 days at 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 status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0117] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0118] After testing, the yield of cyclopentadiene ether terpenoid compounds reached 13.5 mg / g dry cell dry weight, and the purity was 73%.

[0119] Comparative Example 5

[0120] The method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds comprises the following steps:

[0121] (1) Preparation of chitosan oligosaccharide solution: chitosan oligosaccharide with a molecular weight of 2000 Da was selected and dissolved in a sterilized phosphate buffer with a pH value of 6.0 to prepare a chitosan oligosaccharide 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 make it contain 0.3 mg / L naphthaleneacetic acid and 1.2 mg / L 6-benzylaminopurine;

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

[0124] (2) inoculating the Rehmannia glutinosa cambium meristem stem cells into the first modified MS medium and culturing them to the logarithmic growth phase;

[0125] (3) Stem cells in the logarithmic growth phase were immersed in a chitosan oligosaccharide solution for 3 hours by immersion, and then washed with sterile water; then transferred to the second modified MS medium and cultured for 11 days at 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 status of the stem cells was regularly observed, and the cell morphology and proliferation were examined under a microscope to ensure their healthy growth. If contamination or abnormal growth was found, appropriate treatment measures were taken in a timely manner.

[0126] (4) Product extraction: After the culture, the stem cells were collected and the cyclopentadiene ether terpenoid compounds were extracted by ultrasound-assisted organic solvent extraction with ethanol as the solvent (solid-liquid ratio 1 g:10 mL), ultrasound power of 200 W, and ultrasound time of 20 min.

[0127] After testing, the yield of cyclopentadiene ether terpenoid compounds reached 18 mg / g dry cell dry weight, and the purity was 81%.

[0128] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for inducing Rehmannia glutinosa stem cells to produce terpenoid compounds, characterized in that: The following steps are involved: (1) preparing chitosan oligosaccharide solution; Adding auxin and cytokinin to a basic MS medium to prepare a first improved MS medium; increasing the nitrogen source concentration in the first improved MS medium by 5%-25%, decreasing the phosphorus source concentration by 3%-15%, increasing the potassium source concentration by 3%-15%, and adding a plant peptide with a concentration of 0.005-0.1 mg / L and a β-cyclodextrin with a concentration of 0.5%-6% (w / v) to prepare a second improved MS medium; (2) inoculating the Rehmannia stem cells into the first modified MS culture medium and culturing them to the logarithmic growth phase; then soaking the Rehmannia stem cells in the logarithmic growth phase in a chitosan oligosaccharide solution for 0.5-5 hours; then transferring them to the second modified MS culture medium and culturing them under light; after the culturing, collecting the Rehmannia stem cells and extracting terpenoid compounds.

2. The method according to claim 1, characterized in that The process of preparing the chitosan oligosaccharide solution is as follows: selecting chitosan oligosaccharide with a molecular weight of 800-4000Da, dissolving it 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).

3. The method according to claim 1 or 2, characterized in that: In the first improved 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.

4. The method according to claim 3, characterized in that In the first improved 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.

5. The method according to claim 4, characterized in that The auxin is naphthaleneacetic acid; the cytokinin is 6-benzylaminopurine.

6. The method according to claim 1 or 2, characterized in that: In step (1), the nitrogen source concentration in the first improved MS medium is increased by 10%-20%, the phosphorus source concentration is reduced by 5%-10%, the potassium source concentration is increased by 5%-10%, and a plant peptide with a concentration of 0.01-0.1 mg / L and a β-cyclodextrin with a concentration of 1%-5% (w / v) are added to prepare a second improved MS medium.

7. The method according to claim 1 or 2, characterized in that: In step (2), when the Rehmannia stem cells are inoculated in the first modified MS culture medium, each milliliter of the first modified MS culture medium contains 1×10 4 -5×10 4 stem cells.

8. The method according to claim 1 or 2, characterized in that: In step (2), the culture process is: culture for 6-15 days under the conditions of a temperature of 23-30°C, a light intensity of 1600-2800 lux, and a light duration of 8-15 hours / day.

9. The method according to claim 1 or 2, characterized in that: In step (2), the Rehmannia stem cells are immersed in the chitosan oligosaccharide solution for 1-4 hours.

10. The method according to claim 1, characterized in that In step (2), the terpene compound is extracted by ultrasonic-assisted organic solvent extraction.