A regulatory method for simultaneously increasing the total flavonoids and various organic acids in the callus of Leontopodium alpinum

Abstract

The present invention discloses a regulation method for simultaneously enhancing the total flavonoids and various organic acids in the callus and suspension-cultured cells of *Leontopodium alpinum*. The present invention mainly adopts a specific regulation scheme, and simultaneously adds two kinds of inducers with excellent synergistic effects, an AgNO 3 aqueous solution and a 95% ethanol solution of salicylic acid (SA), which can improve the contents of total flavonoids, chlorogenic acid, and leontopodic acid in the callus and suspension cells of *Leontopodium alpinum*. According to the cultivation strategy described in this patent, the cell line of *Leontopodium alpinum* can maintain stable growth and the expression of flavonoid and various organic acid secondary metabolites at a scale of 0.1 - 200 L.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and relates to a regulation method for simultaneously increasing the total flavonoids and various organic acids in the callus of Leontopodium alpinum. Background Art

[0002] Leontopodium alpinum is a plant of the genus Leontopodium in the family Asteraceae, also known as "edelweiss", a perennial herb, a famous alpine flower in Europe, listed as an endangered species by the International Union for Conservation of Nature, and listed as a protected plant in many countries.

[0003] Leontopodium alpinum has a high self-defense ability and can resist bad weather and other growth environments. Through research, it is found that Leontopodium alpinum contains a variety of chemical components. So far, the isolated ones include terpenoids, phenylpropanoids (phenolic acids, flavonoids, coumarins, lignans), fatty acids, polyacetylenes, etc. Among them, the underground part of Leontopodium alpinum contains more sesquiterpenoids, lignans and polyacetylenes, while the above-ground part contains more flavonoids, phenolic acids (chlorogenic acid, leontopodic acid, leontopodic acid B) and fatty acids. In the early growth stage of Leontopodium alpinum, there is almost no synthesis of substances such as flavonoids. After flowering until the full-bloom stage, various metabolites begin to accumulate, but the content of flavonoid compounds is still at a relatively low level; leontopodic acid is considered a natural antioxidant, with a structure similar to chlorogenic acid, and is an organic acid with antioxidant activity, which exists in the above-ground part of Leontopodium alpinum at the full-bloom stage. Compared with other natural antioxidants, the effect of leontopodic acid is more mild. Leontopodium alpinum not only has good pharmacological activities in the cardiovascular and nervous systems, but also has anti-inflammatory, antibacterial, antioxidant, diuretic and chemical prevention effects.

[0004] At present, there is no patent on the metabolic regulation of Leontopodium alpinum callus, and there is no literature report on the large-scale regulation of Leontopodium alpinum callus. Most metabolic regulation methods only target one type of target compound (such as flavonoids), and there is no report on the metabolic regulation method of Leontopodium alpinum that can simultaneously increase the content of two types of active ingredients. Summary of the Invention

[0005] The purpose of this patent is to fill the technical gap in the prior art, and provide a regulation method for Leontopodium alpinum callus that can simultaneously induce the synthesis of flavonoid compounds and various organic acids, which can stably increase the yield of active ingredients in shake flask and disposable reactor scale-up culture, enhance the efficacy of Leontopodium alpinum cells, and reduce the production cost of various key ingredients.

[0006] To achieve the above purpose, the present invention discloses the following technical content:

[0007] A regulatory method for simultaneously increasing the total flavonoids and various organic acids in the callus and suspension-cultured cells of Leontopodium alpinum, which is characterized by the following steps:

[0008] (1) Suspension culture of Leontopodium alpinum callus: Select the Leontopodium alpinum callus with loose texture, light yellow to yellow color, and fast growth rate, and inoculate it into the suspension medium CM2 at a ratio of 0.1 - 2 g / 10 mL. Place it on a shaker and culture at 100 - 115 rpm, keep the temperature at 20 - 25 °C, and subculture it every 7 - 10 d at a ratio of 5 - 15 g / 100 mL (wet weight). The growth rate is 2 - 3 times / 7 d. The suspension medium CM2 mentioned above refers to: B5 + 1 - 2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 1 - 2 mg / L 2,4-D + 0.1 - 0.2 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8 - 6.0;

[0009] (2) One-time bioreactor culture of Leontopodium alpinum callus: Select the suspension-cultured materials that have been cultured for 7 d with consistent state, and inoculate 1500 - 2500 mL of the culture and 2500 - 3500 mL of the sterilized suspension medium CM2 into a 5 L one-time bioreactor to make the final volume 5000 mL. The vibration frequency is 9.5 times / min, and the air supply is 0.1 m³ / h. Culture for 7 d. When subculturing, inoculate the culture into a new one-time bioreactor, add 2 - 4 times the volume of the suspension medium CM2, and culture with oscillation and aeration. The oscillation frequency range is 2 - 15 times / min, and the aeration volume is 0.05 - 0.3 m³ / h;

[0010] (3) Metabolic regulation process: Sterilely add an aqueous solution of 50 mM AgNO 3 filtered through a 0.22 μm microporous membrane and a 95% ethanol solution of 500 mM salicylic acid (SA) to the Leontopodium alpinum suspension culture that has grown for 5 - 7 days, so that their final concentrations reach 2.5 - 15 μM (AgNO 3 ) and 20 - 100 mM (SA) respectively; continue to culture according to the original culture protocol for 2 - 5 days and then harvest.

[0011] The present invention further discloses the application of the method in simultaneously increasing the contents of total flavonoids, chlorogenic acid, and leontopodic acid in the callus and suspension-cultured cells of Leontopodium alpinum. The experimental results show that: according to the culture strategy described in this patent, the Leontopodium alpinum cell line can maintain stable growth and the expression of flavonoid and various organic acid secondary metabolites at a scale of 0.1 - 200 L (total flavonoids ≥ 15%, chlorogenic acid ≥ 2%, leontopodic acid ≥ 1.4%, dicaffeoylquinic acid ≥ 4%).

[0012] A more detailed description of the present invention is as follows:

[0013] Suspension culture of Leontopodium alpinum callus: Select Leontopodium alpinum callus with loose texture, light yellow to yellow color, and fast growth rate, and inoculate it into suspension medium CM2 (B5 + 1 - 2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 1 - 2 mg / L 2,4-D + 0.1 - 0.2 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8 - 6.0) at a ratio of 0.1 - 2 g / 10 mL, place it on a shaker and culture at 100 - 115 rpm, keep the temperature at 20 - 25°C, subculture every 7 - 10 d at a ratio of 5 - 15 g / 100 mL (wet weight), and the growth rate is 2 - 3 times / 7 d.

[0014] One-time bioreactor culture of Leontopodium alpinum callus: Select suspension culture materials that have been cultured for 7 d with consistent states, inoculate 1500 - 2500 mL of the culture and 2500 - 3500 mL of sterilized suspension medium CM2 into a 5 L one-time bioreactor to make the final volume 5000 mL, the vibration frequency is 9.5 times / min, the air flow is 0.1 m³ / h, and culture for 7 d. When subculturing, inoculate the culture into a new one-time bioreactor, add 2 - 4 times the volume of suspension medium CM2, oscillate and aerate for culture, the oscillation frequency range is 2 - 15 times / min, and the air flow rate is 0.05 - 0.3 m³ / h. The culture is carried out in a bioreactor disclosed in Chinese patent documents with publication numbers CN103224882A or CN204385208U, and the vibration frequency of the reactor during the culture process is 5 - 20 times / min.

[0015] Metabolic regulation process: Sterilely add 50 mM AgNO filtered through a 0.22 μm microporous membrane to the Leontopodium alpinum suspension culture that has grown for 5 - 7 days 3An aqueous solution and a 95% ethanol solution of 500 mM salicylic acid (SA) were added to make their final concentrations reach 2.5 - 15 μM (AgNO 3 ), and 20 - 100 mM (SA), respectively. The culture was continued according to the original culture protocol for 2 - 5 days and then harvested. At this time, the total flavonoid content of the callus could be increased to 145% - 190%, the chlorogenic acid content was increased to 135% - 187%, the leontolic acid was increased to 156% - 193%, and the dicaffeoylquinic acid was increased to 115% - 190%.

[0016] By adopting a specific regulation scheme (simultaneously adding two kinds of inducers with excellent synergistic effects), the contents of total flavonoids, chlorogenic acid, leontolic acid, and dicaffeoylquinic acid in the callus of Leontopodium alpinum can be increased.

[0017] The key points of the regulation method disclosed in the present invention for simultaneously increasing the total flavonoids and various organic acids in the callus and suspension-cultured cells of Leontopodium alpinum, which are different from the existing schemes, are as follows:

[0018] Currently, there is no patent on the metabolic regulation of the callus of Leontopodium alpinum, and there is no publicly reported literature on the large-scale regulation of the callus of Leontopodium alpinum. Most metabolic regulation methods only target one type of target compound (such as flavonoids), and there is no reported metabolic regulation method for Leontopodium alpinum that can simultaneously increase the contents of two types of active ingredients. Through a large number of inducer screenings and analysis of synergistic effects, this patent has obtained a regulation scheme that can simultaneously increase the flavonoid compounds and organic acids in the cell line. By using the regulation method described in this patent, the metabolic regulation of the callus of Leontopodium alpinum with different culture scales can be achieved. Description of the Drawings

[0019] Figure 1 It is a process flow chart of the regulation method for simultaneously increasing the total flavonoids and various organic acids in the callus and suspension-cultured cells of Leontopodium alpinum. Detailed Embodiments

[0020] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well-known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples without departing from the essence and scope of the present invention also belong to the protection scope of the present invention. Seeds of Leontopodium alpinum are commercially available, and the raw materials and reagents used in the present invention are also commercially available.

[0021] Example 1:

[0022] Flask culture and regulation of the callus of Leontopodium alpinum

[0023] Select the callus of *Leontopodium alpinum* with loose texture, light yellow color, and fast growth rate, and inoculate it into the high-temperature sterilized suspension medium CM2 (B5 + 1 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8 - 6.0) at a ratio of 1 g / 10 mL. Place it on a shaker and culture at 105 rpm, keep the temperature at 24°C. After the biomass reaches 12 g, inoculate it into a 500 mL shake flask (the medium volume is 100 mL) at a ratio of 12 g / 100 mL (wet weight), and subculture it every 7 days. After 5 subcultures, on the 6th day of the 6th growth cycle, add 20 μL of 50 mM silver nitrate mother liquor filtered through a 0.22 μm microporous filter membrane and 20 μL of 500 mM salicylic acid mother liquor to the suspension culture. Use the sample with an equal amount of sterile water added as the control group, place it in the original culture conditions and continue to culture for 3 days, harvest the cells, and dry them at 65°C. 3 The cells of *Leontopodium alpinum* after drying were taken to detect the contents of total flavonoids and various organic acids. The detection methods are as follows:

[0024] Take the dried cells of *Leontopodium alpinum* to detect the contents of total flavonoids and various organic acids. The detection methods are as follows:

[0025] (1) Detection of total flavonoid content:

[0026] Grind and crush the dried cells of *Leontopodium alpinum*, and pass through a 40-mesh sieve to obtain dry powder; weigh 0.1000 g of dry powder into a 50 mL centrifuge tube, add 25 mL of 80% ethanol, and mix well with a vortex mixer; sonicate in an ultrasonic cleaner for 30 min (power 500 W, temperature 60°C); centrifuge at 5000 rpm / min for 10 min, and measure the total flavonoid content of the supernatant. Take 6 mL of the test solution, place it in a 25 mL volumetric flask, add 1 mL of 5% sodium nitrite solution, shake well, let stand for 6 minutes, add 1 mL of 10% aluminum nitrate solution, shake well, let stand for 6 minutes, add 10 mL of 4% sodium hydroxide test solution, dilute to the scale with pure water, shake well, and let stand for 15 min. Use the corresponding reagent as the blank, and according to the spectrophotometry method (Appendix IV A, Part II of the Chinese Pharmacopoeia 2010 Edition), measure the absorbance at a wavelength of 500 nm. Use rutin as the reference substance, and calculate the parameters of the linear regression equation y = kx + b with the absorbance and its corresponding mass. Calculate the total flavonoid content (calculated as rutin) of the sample according to the formula C = (A - b) × V 0 / (k × m × V 1 ) where: C - the content of total flavonoids in the sample, mg / g; A - the absorbance value measured for the sample; k, b - the slope and intercept in the standard curve; m - the mass of the sample taken; V 0 -- the total volume after the sample extract is diluted to volume; V 1 -- the volume taken for measurement.

[0027] (2) Detection of organic acid content

[0028] Grind and crush the dried Leontopodium alpinum cells, sieve through a 40-mesh sieve to obtain dry powder; weigh 0.1 g of the sample, add 25 mL of 80% ethanol, sonicate for 30 min, centrifuge (5000 rpm, 10 min), take 200 μL of the supernatant and place it in the liner of a UPLC injection vial for testing. The detection conditions are as follows:

[0029] Chromatographic column: Agela Vensil ASB C18 3 μm 150 Å 2.1×100 mm 4# chromatographic column

[0030] Mobile phase: C: methanol; D: 0.2% phosphoric acid aqueous solution, perform gradient elution according to the following table

[0031]

[0032] Column temperature: 40 °C; Sample temperature: 10 °C; Flow rate: 0.5 ml / min

[0033] Detection wavelength: 327 nm (the maximum absorption wavelength of chlorogenic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, leontolic acid A,

[0034] leontolic acid B)

[0035] Take 1 μL of the test solution, inject it into the UPLC, measure, and inject samples in parallel for 2 needles. Record the chromatogram for 16 minutes and the chromatographic peak areas of chlorogenic acid, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, leontolic acid A, and leontolic acid B. Calculate the content according to the standard curve using the average peak area of the 2 needles.

[0036] The detection results are as follows:

[0037] Table 1 Content of secondary metabolites in the cells of Leontopodium alpinum cultured and regulated in shake flasks

[0038]

[0039] Example 2

[0040] One-time bioreactor culture and regulation of Leontopodium alpinum callus

[0041] Suspension culture of Leontopodium alpinum callus: Select the Leontopodium alpinum callus with loose texture, light yellow color, and fast growth rate, and inoculate it into the sterilized suspension medium CM2 (B5 + 2 mg / L 2,4-D + 0.2 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8 - 6.0) at a ratio of 1 g / 10 mL. Place it on a shaker and culture at 105 rpm, keep the temperature at 24°C. After the biomass reaches 12 g, inoculate it into a 500 mL shake flask (the medium volume is 100 mL) at a ratio of 12 g / 100 mL (wet weight), and subculture once every 7 days.

[0042] Culture in a 5-L disposable bioreactor: After 7 generations of shake flask culture, inoculate 1500 mL of suspension culture and 2000 mL of sterilized suspension medium CM2 into a 5-L disposable bioreactor, and culture it in the bioreactor disclosed in the Chinese patent documents with publication numbers CN103224882A or CN204385208U. The vibration frequency is 9.5 times / min, the air flow rate is 0.1 m³ / h, and culture for 7 days.

[0043] Subculture in a 20-L disposable bioreactor: Inoculate 5000 mL of suspension culture into a 20-L disposable bioreactor, add 7000 mL of sterilized suspension medium CM2, and culture it in the bioreactor disclosed in the Chinese patent documents with publication numbers CN103224882A or CN204385208U. The vibration frequency is 9.5 times / min, the air flow rate is 0.2 m³ / h, and culture for 7 days.

[0044] Subculture in a 100 - 200-L disposable bioreactor: Inoculate 40 L of suspension culture into a 100 - 200-L disposable bioreactor, add 60 L of sterilized suspension medium CM2, and culture it in the bioreactor disclosed in the Chinese patent documents with publication numbers CN103224882A or CN204385208U. The vibration frequency is 5.5 times / min, the air flow rate is 0.3 m³ / h, the oxygen flow rate (95% purity) is 0.06 m³ / h. On the 5th day of culture, add 25 mL of 50 mM AgNO 3 Mother liquor and 40 mL of 500 mM salicylic acid mother liquor after filtration sterilization. Use the sample added with an equal amount of sterile water as the control group, and continue to culture for 3 days under the original culture conditions to harvest fresh cells.

[0045] Take the dried Leontopodium alpinum cells to detect the contents of total flavonoids and various organic acids. The detection method refers to the method in Example 1, and the detection results are as follows:

[0046]

[0047] Example 3

[0048] Comparative experiment

[0049] Using the method of the present invention, the total flavonoids and various organic acids in the callus culture of *Leontopodium alpinum* are significantly increased. Among them, the content of total flavonoids can be increased to 145%-190%, the content of chlorogenic acid can be increased to 135%-187%, the content of leontolic acid can be increased to 156%-193%, and the content of dicaffeoylquinic acid can be increased to 115%-190%.

[0050]

[0051] Conclusion: The method of the present invention can efficiently obtain the tissue culture of *Leontopodium alpinum* rich in flavonoids and various organic acids (total flavonoids ≥ 15%, chlorogenic acid ≥ 2%, leontolic acid ≥ 1.4%, dicaffeoylquinic acid ≥ 4%). According to the culture strategy described in this patent, the cell line of *Leontopodium alpinum* can maintain stable growth and the expression of secondary metabolites of flavonoids and various organic acids at a scale of 0.1-200 L.

Claims

1. A regulatory method for simultaneously increasing the total flavonoids and various organic acids in the callus and suspension-cultured cells of Leontopodium alpinum. The organic acids refer to chlorogenic acid, leontopodic acid, and dicaffeoylquinic acid. It is characterized in that it is carried out according to the following steps: (1) Suspension culture of Leontopodium alpinum callus: Select Leontopodium alpinum callus with loose texture, light yellow to yellow color, and fast growth rate. Inoculate it into suspension medium CM2 at a ratio of 0.1 - 2 g / 10 mL, place it on a shaker and culture at 100 - 115 rpm, keep the temperature at 20 - 25 °C, subculture every 7 days at a ratio of 5 - 15 g / 100 mL wet weight, and the growth rate is 2 - 3 times / 7 d. The suspension medium CM2 refers to: B5 + 1 - 2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 1 - 2 mg / L 2,4-D + 0.1 - 0.2 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8 - 6.0; (2)Metabolic regulation process: Sterilely add an aqueous solution of 50 mM AgNO filtered through a 0.22 μm microporous filter membrane and 500 mM salicylic acid SA, 95% ethanol solution to the suspension culture of Leontopodium alpinum that has grown for 7 days, so that their final concentrations reach 2.5 - 15 μM AgNO 3 and 20 - 100 mM SA respectively; continue to culture according to the original culture protocol for 2 - 5 days and then harvest; 3 ​ Or (1) Suspension culture of Leontopodium alpinum callus: Select Leontopodium alpinum callus with loose texture, light yellow to yellow color, and fast growth rate. Inoculate it into suspension medium CM2 at a ratio of 0.1 - 2 g / 10 mL, place it on a shaker and culture at 100 - 115 rpm, keep the temperature at 20 - 25 °C, subculture every 7 days at a ratio of 5 - 15 g / 100 mL wet weight, and the growth rate is 2 - 3 times / 7 d. The suspension medium CM2 refers to: B5 + 1 - 2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 1 - 2 mg / L 2,4-D + 0.1 - 0.2 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, pH 5.8 - 6.0; (2) Submerged culture of Leontopodium alpinum callus in a disposable bioreactor: Select suspension culture materials that have been cultured for 7 days with consistent status. Inoculate 1500 - 2500 mL of the culture and 2500 - 3500 mL of sterilized suspension medium CM2 into a 5 L disposable bioreactor to make the final volume 5000 mL. The oscillation frequency is 9.5 times / min, and the air flow rate is 0.1 m³ / h. Culture for 7 days. When subculturing, transfer the culture into a new disposable bioreactor, add 2 - 4 times the volume of suspension medium CM2, and culture with oscillation and aeration. The oscillation frequency range is 2 - 15 times / min, and the aeration volume is 0.05 - 0.3 m³ / h. The suspension medium CM2 mentioned above refers to: B5 + 1 - 2 mg / L 2,4-D + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein or B5 + 1 - 2 mg / L 2,4-D + 0.1 - 0.2 mg / L 6-BA + 30 g / L sucrose + 0.1 mg / L L-glutamine + 0.2 mg / L hydrolyzed milk protein, with a pH of 5.8 - 6.0; (3)Metabolic regulation process: Sterilely add 50 mM AgNO aqueous solution filtered and sterilized with a 0.22 μm microporous filter membrane and 500 mM salicylic acid SA, 95% ethanol solution to the suspension culture of Leontopodium alpinum that has grown for 7 days, so that their final concentrations reach 2.5 - 15 μM for AgNO and 20 - 100 mM for SA respectively; continue to culture according to the original culture protocol for 2 - 5 days and then harvest. 3 3 ​​ 2. Use of the method for regulating the total flavonoids and various organic acids in Leontopodium alpinum callus and suspension culture cells as claimed in claim 1 in increasing the contents of total flavonoids, chlorogenic acid, and leontopodic acid in Leontopodium alpinum callus and suspension culture cells.

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