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.

Inactive Publication Date: 2011-04-20
SINOBIOWAY BIO AGRI GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few studies on the content, influencing factors and how to use flavones in the hairy root culture solution. Although Yu Shuhong et al. reported the effect of Tween-80 on the content of isoflavones in kudzu hairy roots, But it does not involve the research on licorice, let alone the research and enlightenment on the content of flavone (flavone) in the hairy root of licorice and its culture solution
In addition, Tween, as a commonly used pharmaceutical adjuvant, can be used as an adjuvant in traditional Chinese medicine compound preparations to form a pharmaceutical composition together with active ingredients such as licorice or its extracts, such as Chinese patents 95108283, 02100222, and 200610035831, etc., but in In these drugs, Tween exists only as a pharmaceutical adjuvant

Method used

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  • Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof
  • Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof
  • Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof

Examples

Experimental program
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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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Abstract

The invention provides a culture method of glycyrrhiza hairy roots and a method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof. The method comprises the following steps: culturing glycyrrhiza hairy roots in a fluid nutrient medium which does not contain Tween to the growth peak period; culturing the obtained glycyrrhiza hairy roots in the growth peak period in a fluid nutrient medium which contains Tween for more than 5 days; and eluting by ethanol with different concentration through a polyamide chromatography column, and separating the total flavonoids from the culture solution, wherein the transfer rate of the glycyrrhiza total flavonoids is 97.23%, the transfer rate of glycyrrhiza chalcone A is 98.14%, and the transfer rate of Glabridin is 97.54%. The invention also provides the glycyrrhiza total flavonoids prepared by the method and medical applications of the glycyrrhiza total flavonoids.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for cultivating licorice hairy roots and a method for preparing licorice total flavonoids by using licorice hairy roots and their culture solution. In addition, the present invention also relates to a total flavonoids of licorice obtained by the above preparation method and its medical application. Background technique [0002] Licorice is an important traditional Chinese medicine in my country. It has the functions of clearing heat and detoxifying, moisturizing the lungs and eliminating phlegm, relieving spasms and relieving pain, invigorating qi and benefiting the spleen and stomach, and harmonizing various medicines. It is known as the "king of all herbs". In recent years, it has been found that various flavonoids contained in licorice are another type of active substance besides glycyrrhizic acid. The total flavonoids of licorice have anti-oxidatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04A61K36/484A61P31/18A61P29/00A61P35/00A61P39/06
Inventor 詹姆斯·周高春春刘敬梅陈春燕李毅
Owner SINOBIOWAY BIO AGRI GRP CO LTD
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