Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof
A technology of licorice total flavonoids and hairy roots, applied in the biological field, can solve problems that do not involve licorice research, flavonoids, etc.
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Embodiment 1
[0049] Embodiment 1, the cultivation of Radix Glycyrrhizae hairy root
[0050] Soak fresh Ural licorice seeds (available from Bozhou Medicinal Materials Corporation) with 70% (v / v) ethanol for 10 seconds, rinse with sterile water for 3 times, and then soak with 2% (w / w) sodium hypochlorite solution for 10 seconds. Minutes, washed 3 times with sterile water, and then inoculated the seeds on MS solid medium (that is, MS liquid medium with 1% (w / w) agar added), cultured at 25°C in the dark for 4 days, and the roots Cut the middle into 0.5cm long pieces.
[0051] Transfer the subsection to MS solid culture supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (2 mg / L final concentration) and kinetin (KT) (0.2 mg / L final concentration) culture medium at 25°C in the dark for 40 days to obtain villous adventitious roots (ie, hairy roots).
[0052] Then, the hairy roots were transferred to MS liquid medium and cultured for 20 days at 25° C. in the dark on a shaker at 100 rpm. Af...
Embodiment 2
[0054] Embodiment 2, preparation of total flavonoids in Glycyrrhizae hairy root
[0055]Take 150 g of licorice hairy roots induced by Tween culture prepared according to Example 1, place them in an oven, and dry at 60° C. for 12 hours to obtain 10 g of dried hairy roots. Put the hairy roots in a 500mL round-bottomed flask, add 200mL of water, soak at 85°C for 7 hours, cool to room temperature, filter with filter paper, discard the filtrate, wash with 60°C warm water three times, 200mL each time, and then The hairy root residue retained by filtration was placed in an oven and dried at 60° C. for 12 hours. Put the dried licorice hairy root residue in a 500mL round bottom flask, add 200mL of 95% (v / v) ethanol, reflux at 85°C for 2 hours, cool to room temperature, filter through filter paper, and keep the filtrate. The filter residue was washed twice with 10 mL of 95% (v / v) ethanol, and the washing liquid was retained. Then, 200 mL of 95% (v / v) ethanol was added to the residue, r...
Embodiment 3
[0058] Embodiment 3, preparation of total flavonoids in licorice hairy root culture solution
[0059] (1) Concentration of licorice hairy root culture solution: 60 mL of the licorice hairy root culture solution induced by Tween-80 prepared according to Example 1 was concentrated to 1 / 2 of the original volume (ie 30 mL) at 60°C.
[0060] (2) Pretreatment of polyamide resin: 100 g of polyamide resin used for column chromatography (available from Sinopharm Chemical Reagent Company) was refluxed at 85° C. for 3 hours with 95% (v / v) ethanol 500 ml, and filtered through filter cloth , then add 500mL of 95% (v / v) ethanol, reflux once at 85°C, and filter through filter cloth to remove residual alcohol-soluble impurities in the polyamide resin. Pack the treated polyamide resin into a column so that the column volume is 60 mL, and wash the chromatography column with 300 mL of 70% (v / v) ethanol, 50% (v / v) ethanol and 30% (v / v) ethanol successively, Then rinse the chromatography column w...
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