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

[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).