Method for quickly separating and preparing natural dihydroflavonoid compounds by using high-speed counter-current chromatography

A high-speed countercurrent chromatography and dihydroflavonoid technology is applied in the field of preparing dihydroflavonoids by a high-speed countercurrent chromatography continuous cycle separation method, which can solve the problems of long time consumption, single compound structure, and difficulty in one-time separation, etc. high purity effect

Active Publication Date: 2018-02-02
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AI-Extracted Technical Summary

Problems solved by technology

It has been reported in the literature that flavonoids, such as diosmin, luteolin, and 5,7-dihydroxychromanone, were isolated and purified from peanut shells using conventional HSCCC technology (Niu Dandan et al., Natural Product Research and Development, 2011,23:1...
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The invention belongs to the technical field of medicine, and relates to a method for separating and preparing natural dihydroflavonoid compounds. The method adopts a continuous circulation method ofhigh-speed counter-current chromatography, and a two-phase solvent system which is formed by n-hexane, ethyl acetate, methanol and water is adopted in the high-speed counter-current chromatography toseparate and prepare the dihydroflavonoid compounds from roots of sophora alopecuroide. According to the method, a mode of high-speed counter-current circulating elution, an online storage technique and a reverse elution mode are adopted at the same time, and continuous circulating elution are conducted on flow components which contain the target compound to collect the flow components and obtainflavonoid compounds which comprise sophoratonkin, alopecurone F, lehmannin, alopecurone A, sophora flavanone G and alopecurone B and have the purity of more than 95%; the five dihydroflavonoid compounds and santilin glycoside which have high purity are obtained through one-time separation, the method is simple, convenient and rapid in operation, and no loss of the target compounds is caused by material adsorption during the separation.

Application Domain

Sugar derivativesSugar derivatives preparation

Technology Topic

FlavanoneCounter current +10


  • Method for quickly separating and preparing natural dihydroflavonoid compounds by using high-speed counter-current chromatography
  • Method for quickly separating and preparing natural dihydroflavonoid compounds by using high-speed counter-current chromatography
  • Method for quickly separating and preparing natural dihydroflavonoid compounds by using high-speed counter-current chromatography


  • Experimental program(1)

Example Embodiment

[0032] Example 1
[0033] (1) The OptiChrome-300PLUS high-speed counter-current chromatograph produced by Jiangsu Jiangyin Countercurrent Technology Co., Ltd. was used to improve sample separation and purification, and the Agilent 1200HPLC high-performance liquid chromatograph produced by Agilent Co., Ltd. was used for sample purity analysis;
[0034] Set up the high-speed countercurrent chromatograph, connect the six-way valve a after the instrument detector, connect the six-way valve a to the storage ring, and connect the six-way valve b before the constant flow pump, which is responsible for switching between normal elution and cyclic elution ( Such as figure 1 Shown);
[0035] (2) Preparation of extract samples
[0036] Take the sophora sphaerocarpa root, dry the root, and grind it, cold soak and extract three times with 95% ethanol, combine the extracts, concentrate under reduced pressure, and evaporate to dryness;
[0037] (3) Prepare a two-phase solvent system: mix n-hexane, ethyl acetate, methanol and water in a ratio of 9:6:6~8:6~8 (v:v), and then stand for separation, respectively Take the upper and lower phases and ultrasonically degas for 20 minutes to remove bubbles;
[0038] (4) Preparation of sample solution: Dissolve 200 mg of the above-mentioned Sophora flavescens root extract in each 5 mL upper and lower phase solvent system;
[0039] (5) High-speed countercurrent chromatographic separation: Pump the stationary phase into the chromatographic column at a flow rate of 30 mL/min. After the stationary phase fills the entire chromatographic column, start the host and set the rotation speed to 1200 rpm. The phase is pumped into the chromatographic column at a flow rate of 15 mL/min; after the two-phase system reaches dynamic equilibrium in the chromatographic column, 10 mL of sample solution is injected into the chromatographic column through the six-way injection valve; the flow is controlled by the six-way valve a A is stored in the storage ring; after fraction B is collected, the instrument enters the circulation separation mode by controlling the six-way valves a and b, and fraction A is collected after 3 cycles of separation; after fraction A is completely eluted , By controlling the switch, change the mobile phase to the upper phase, control the instrument to enter the reverse elution mode (end-to-end), collect fraction C; use UV detector to detect the fraction during the separation process, the wavelength is 254nm, and the chromatography workstation The data is collected and processed, and the fractions are collected by an automatic fraction receiver at the same time, 10 mL per tube; the high-speed countercurrent chromatogram obtained in the experiment is as follows figure 2 As shown, it is shown that five dihydroflavonoids and one pterolin monomer (compounds 1-6) are well separated;
[0040] (6) Sample combination and purity analysis: use high performance liquid chromatography to detect the purity of the collected fractions; the chromatographic separation conditions are: Eclipse XDB-C18column (5μm, 250mm×4.6mm, id), column temperature 25 ℃, the mobile phase is acetonitrile: water=55:45, the flow rate is 1mL/min, and the detection wavelength is 254nm; combine the fractions with higher purity and the same retention time, use a rotary evaporator to remove the solvent in the combined fractions to obtain Formula (I, II, III, IV shown in the dihydroflavonoid monomer fractions, high performance liquid chromatography analysis of the final separated dihydroflavonoids Sophoratonkin, Alopecurone F, Lehmannin, Alopecurone A, Sophora flavanone G and Alopecurone B;. The purity is greater than 95% (such as image 3 Shown).


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