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Regulation and control method for simultaneously increasing general flavones and multiple organic acids of leontopodium alpinum calluses

A technology of alpine edelweiss and callus, applied in the field of plant tissue culture, to achieve the effect of increasing the content

Pending Publication Date: 2019-04-09
TIANJIN ACELBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most metabolic regulation methods only target one type of target compounds (such as flavonoids), and there is no report on the metabolic regulation method of alpine edelweiss that can simultaneously increase the content of two types of active ingredients

Method used

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  • Regulation and control method for simultaneously increasing general flavones and multiple organic acids of leontopodium alpinum calluses
  • Regulation and control method for simultaneously increasing general flavones and multiple organic acids of leontopodium alpinum calluses
  • Regulation and control method for simultaneously increasing general flavones and multiple organic acids of leontopodium alpinum calluses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Shake Flask Culture and Regulation of Alpine Edelweiss Callus

[0023] Select alpine edelweiss callus with loose texture, light yellow color and fast growth rate, and insert it into 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), cultured on a shaker at 105 rpm, maintained at 24°C, and press 12 The ratio of g / 100 mL (wet weight) was inserted into a 500 mL shake flask (medium volume is 100 mL), and subcultured every 7 days. After 5 passages, add 50 mM AgNO filtered through a 0.22 μm Millipore filter to the suspension culture on day 6 of the 6th growth cycle 3 20 μL of the mother solution and 20 μL of the 500 mM salicylic acid mother solution were used as the control group, and the samples added with the same amount of sterile water were used as the control group. The cells were cultured under the original culture conditions for 3 days, and the cells were harvest...

Embodiment 2

[0040] Disposable bioreactor culture and regulation of callus of Edelweiss alpine

[0041] Suspension culture of alpine edelweiss callus: select alpine edelweiss callus with loose texture, light yellow color and fast growth rate, and insert it into high-temperature sterilized suspension medium CM2 (B5 + 2 mg / L 2,4-D + 0.2mg / 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), placed in a shaker at 105 Rpm culture, the temperature was maintained at 24°C, after the biomass reached 12 g, it was transferred into a 500 mL shake flask (medium volume: 100 mL) at a ratio of 12 g / 100 mL (wet weight), and subcultured every 7 days.

[0042] 5-liter scale disposable bioreactor cultivation: After 7 generations of shake flask cultivation, insert 1500 mL of suspension culture and 2000 mL of sterilized suspension medium CM2 into the 5-liter disposable bioreactor, the publication number is CN103224882A Or in the bioreactor disclosed in the Chinese pate...

Embodiment 3

[0048] Comparative Test

[0049] Utilize the method of the present invention, the total flavonoids and various organic acids of alpine edelweiss callus culture are significantly improved, wherein the total flavone content can be increased to 145%-190%, and the chlorogenic acid content is increased to 135%- 187%, edelweisic acid increased to 156%-193%, dicaffeoylquinic acid increased to 115%-190%;

[0050]

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

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Abstract

The invention discloses a regulation and control method for simultaneously increasing general flavones and multiple organic acids of leontopodium alpinum calluses and suspension culture cells. According to the method, a specific regulation and control scheme is mainly used, two inducers with excellent synergetic effects are simultaneously used, namely an AgNO3 water solution and a salicylic acid (SA) 95% ethanol solution, and then the contents of general flavones, chlorogenic acid and leontopodium alpinum acid contents of the leontopodium alpinum calluses and the suspension culture cells can be all increased. By adopting the culture strategy of the invention, stable growth of leontopodium alpinum cell systems can be kept at a scale of 0.1-200 L, and expression of flavones and multiple organic acid type secondary metabolites can be maintained.

Description

technical field [0001] The invention belongs to the technical field of plant tissue culture, and relates to a control method for simultaneously increasing total flavonoids and various organic acids in alpine edelweiss callus. Background technique [0002] Alpine edelweiss (Leontopodium alpinum) is a plant of the genus Edelweiss in the Compositae family, also known as "Edelweiss", a perennial herb, a famous alpine flower in Europe, and is listed as an endangered species by the International Union for Conservation of Nature, and is also listed as an endangered species by many countries. protected plants. [0003] Edelweiss has a high degree of self-defense and can resist harsh weather and other growing environments. The alpine edelweiss plant contains a variety of chemical components. So far, terpenoids, phenylpropanoids (phenolic acids, flavonoids, coumarins, lignans), fatty acids, and polyacetylenes have been isolated. . Among them, the underground part of edelweiss conta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12P7/40C12P17/06
CPCC12N5/04C12P7/40C12P17/06
Inventor 王鹏刘水燕何俊雄侯蒙蒙张卫谭光伟
Owner TIANJIN ACELBIO BIOTECH
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