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Method for separating cyclopamine analogs from Veratrum plants by high-speed counter-current chromatography

A high-speed countercurrent chromatography, veratramide technology, applied in the direction of steroids, organic chemistry, etc., can solve the problems of restricting in-depth research on anti-tumor drugs, cumbersome extraction and separation operations, and harm to researchers, and achieves good product purity and simple methods. Fast, efficient separation results

Inactive Publication Date: 2014-01-22
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the content of cyclopamine in plants is low, less than one ten-thousandth, and the conventional extraction and separation operations are cumbersome and the yield is low. Extensive use of toxic reagents such as benzene is harmful to the environment and researchers. To a large extent, it limits the further in-depth research of this promising anti-tumor drug.

Method used

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  • Method for separating cyclopamine analogs from Veratrum plants by high-speed counter-current chromatography
  • Method for separating cyclopamine analogs from Veratrum plants by high-speed counter-current chromatography
  • Method for separating cyclopamine analogs from Veratrum plants by high-speed counter-current chromatography

Examples

Experimental program
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Effect test

Embodiment 1

[0044] 1. Preparation of total alkaloids from Veratrum oxysepalum Turcz.

[0045] Put the commercially available veratrum veratrum in an oven at 60°C for 4 hours, cut off the thin section, and then crush it into a coarse powder (passed through a 20-mesh sieve) with a plant grinder, and store it for later use. Take 100g of crushed medicinal materials, add ammonia water (25%), it is advisable to have just submerged the medicinal materials, alkalize for 1 hour, heat and reflux extraction with 1000mL of 95% ethanol for 3 times (1.5 hours each time), and use a rotary evaporator for the ethanol extract Evaporate to dryness into an extract, then dissolve and filter with 100mL (0.1M) HCl, degrease the filtrate twice with double the amount of petroleum ether, then basify with sodium hydroxide (10%) to pH 9-10, and repeat with chloroform Extracted four times (100mL, 100mL, 80mL, 50mL), combined the chloroform layers, and vacuum dried to obtain 3.4g of veratrum total alkaloid powder, yie...

Embodiment 2

[0057] 1. Preparation of total alkaloids from Veratrum oxysepalum Turcz.

[0058]Put the commercially available veratrum veratrum in an oven at 60°C for 4 hours, cut off the thin section, and then crush it into a coarse powder (passed through a 20-mesh sieve) with a plant grinder, and store it for later use. Take 100g of crushed medicinal materials, add an appropriate amount of ammonia water (25%), it is advisable to submerge the medicinal materials, alkalize for 1 hour, use 1000mL of 70% ethanol for ultrasonic extraction 3 times (1.5 hours each time), and use a rotary evaporator for the ethanol extract Evaporate to dryness into an extract, then dissolve and filter with 100mL (0.1M) HCl, degrease the filtrate twice with double the amount of petroleum ether, then basify with sodium hydroxide (10%) to pH 9-10, and repeat with chloroform Extracted four times (100mL, 100mL, 80mL, 50mL), combined the chloroform layers, and vacuum dried to obtain 2.6g of veratrum total alkaloid powd...

Embodiment 3

[0064] 1. Preparation of Veratrum viridiflorum total alkaloids:

[0065] Take 120 g of dried and pulverized Veratrum veratrum powder (passed through a 20-mesh sieve), prepare total alkaloids according to the method in Example 1, and dry them in vacuum to obtain 0.65 g of total alkaloid powders, with a yield of 0.54%.

[0066] 2. Preparation of cyclopamine analogs by high-speed countercurrent chromatography

[0067] High-speed countercurrent chromatography TBE-300B (Shanghai Tongtian Biotechnology Co., Ltd.) was used to separate and purify mephenamine, veratramine, pseudo-mephenamine and veratrol.

[0068] A. Preparation of Veratramine, Pseudo-Mephenamine and Veratrol in High-Speed ​​Countercurrent Separation

[0069] The two-phase solvent system for primary high-speed countercurrent chromatography is dichloromethane-methanol-water (4:3:2, v / v / v). The methods and steps for the separation and purification, purity detection and structure verification of veratramine, pseudo-ser...

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Abstract

The invention relates to a separation method of a natural drug, and discloses a method for separating cyclopamine analogs from Veratrum plants by high-speed counter-current chromatography. The method is characterized in that Veratrum total alkaloid powder is separated and purified by high-speed counter-current chromatography twice, wherein the solvent system of the first high-speed counter-current chromatography is a mixed solvent composed of dichloromethane, methanol and water; and in the second high-speed counter-current chromatography, the jervine fraction obtained by the first high-speed counter-current chromatography is separated and purified as a reflux sample, and the solvent system is a mixed solvent composed of normal hexane, ethyl acetate, methanol and water. The method is simple and quick, and has the advantages of high recovery rate and high purity of the target compound.

Description

technical field [0001] The invention relates to a method for separating natural medicines, and discloses a method for separating four cyclopamine analogues in plants of the genus Veratrum by using secondary high-speed countercurrent chromatography (HSCCC). Background technique [0002] Cyclopamine is a heterosteroidal alkaloid, mainly found in Liliaceae Veratrum californicum, Veratrum grandiflorum, Cornlily and Fritillaria pallidiflora Schrenk). Studies in the middle of the 20th century found that cyclopamine has teratogenic effects, but studies after the 1990s showed that cyclopamine is a hedgehog signaling pathway inhibitor, because mutations in the hedgehog signaling pathway are associated with the pathogenesis of various tumors, Therefore, cyclopamine, as a potential new anti-tumor drug, has set off a research boom all over the world. In recent years, researchers have carried out more in-depth research on cyclopamine, and believe that it has a better inhibitory effect ...

Claims

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Application Information

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IPC IPC(8): C07J69/00
Inventor 李会军姜艳喻悦
Owner CHINA PHARM UNIV
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