阅读说明：本技术 一种山莴苣素的制备方法 (Preparation method of lactucin ) 是由 阿吉艾克拜尔·艾萨 魏哲洋 孙光映 罗玉琴 于 2020-05-16 设计创作，主要内容包括：本发明涉及一种山莴苣素的制备方法,该制备方法分别采用反相C18制备色谱和葡聚糖LH-20型凝胶色谱,从毛菊苣根提取物中分离得到富含山莴苣素的组分溶液,再通过减压浓缩结晶法得到高纯度的山莴苣素。该方法简单易行制备效率高,可获得纯度高于99%的山莴苣素对照品。所得山莴素对照品可用于药材标准化过程中的定量定性学研究。方法简单易行,纯化效率高,具有一定的应用价值。(The invention relates to a preparation method of lactucin, which adopts reversed phase C18 preparative chromatography and dextran L H-20 type gel chromatography respectively, separates component solution rich in lactucin from extract of hairy chicory root, and obtains high-purity lactucin by reduced pressure concentration crystallization.)

1. The preparation method of lactucin is characterized by comprising the following steps:

a. pulverizing Cichorium intybus Roxb root, extracting with 70% methanol water solution at a ratio of 1:5 under reflux for 1 hr, repeatedly extracting for 3 times, filtering, mixing, and concentrating under reduced pressure to obtain extract; taking the obtained extract, adding water with twice volume, ultrasonically dispersing into suspension, adding petroleum ether or n-hexane with equal volume for degreasing for 3 times, mixing the obtained water phases, adding ethyl acetate with equal volume for extracting for 3 times, mixing the obtained ethyl acetate layers, concentrating under reduced pressure, and freeze-drying under vacuum to obtain a cichorium intybus root extract;

b. b, ultrasonically dissolving 50-2000mg of the cichorium intybus root extract obtained in the step a in a methanol-water solution with the volume ratio of 50m L being 20-100%, centrifuging to remove precipitates, and taking supernatant for later use;

c. preparing a chromatographic column by taking a reversed phase C18, wherein the specification is that the length is 250mm, the inner diameter is 80mm, the particle size of a filling material is 10 mu m, the mass of the filled filler is 800g, connecting the reversed phase pilot test C18 chromatographic column to a preparative chromatograph, adjusting the mobile phase to be a methanol-0.1% formic acid water mixture, wherein the methanol accounts for 20% -65%, the column temperature is controlled to be minus 10 ℃ -50 ℃, balancing the chromatographic column for 10-30min for later use, controlling the flow rate to be 100-180m L/min, collecting the lactucin peak of the extract solution obtained in the step b, carrying out reduced pressure concentration at the temperature of 50 ℃, and carrying out vacuum freeze drying to obtain 5-170mg of lactucin with the purity of 85%;

d. wet packing a glucan L H-20 type gel chromatographic column with the length of 1.5m and the inner diameter of 7cm by using methanol as a column packing reagent, taking 5-170mg of the lactucin with the purity of 85% obtained in the step c, adding 2-3m L methanol for dissolving, loading the lactucin to the glucan L H-20 type gel chromatographic column, washing the glucan L H-20 type gel chromatographic column by using pure methanol as a mobile phase, collecting effluent components to obtain components with the volume of 5m L in each bottle, collecting 20 bottles, respectively measuring the purity of the lactucin in the obtained 20 bottles of components by using a high performance liquid chromatograph, combining the component solutions with the purity of more than 95%, and concentrating by using a rotary evaporator at the temperature of 50 ℃ until the lactucin is dried;

e. concentrating and drying the solid obtained in the step c, adding methanol to dissolve the solid into clear saturated solution at the temperature of 40-70 ℃, placing the solution in a refrigerator at the temperature of 4 ℃ overnight to obtain colorless transparent crystals, filtering the crystals, washing the crystals with petroleum ether to obtain a lactucin reference substance with the purity of 99%, filtering the crystal mother solution to obtain a crystal mother solution, concentrating the crystal mother solution by using a rotary evaporator at the temperature of 50 ℃ to dry the crystal mother solution, adding methanol to dissolve the crystal mother solution into clear saturated solution at the temperature of 40-70 ℃, placing the crystal mother solution in a refrigerator at the temperature of 4 ℃ overnight to obtain colorless transparent crystals, filtering the crystal mother solution, washing the crystals with petroleum ether, and combining the two batches of crystals to obtain the lactucin reference substance with the purity of 99% of 3-105 mg.

2. The method as claimed in claim 1, wherein the amount of the extract of the root of Lactuca sativa in step b is 300-1000mg, and the amount of methanol in the methanol-water used for dissolving the sample of the extract of Lactuca sativa is 50-90%.

3. The method for preparing lactucin according to claim 1, wherein the volume ratio of methanol in the mobile phase is 30-50% and the column temperature is-10-30 ℃ during the purification of the reversed phase C18 preparative chromatography using 800g of filler in step C.

4. A method of preparing lactucin according to claim 1, wherein the crystallization temperature in step e is 60-70 ℃.

Technical Field

The invention relates to a preparation method of lactucin, which takes a cichorium intybus root extract as a raw material, and obtains a lactucin reference substance with the purity higher than 99 percent by combining reversed-phase C18 preparative chromatography, glucan L H-20 column chromatography and a concentration crystallization method.

Background

The method mainly comprises the steps of preparing a lactuca sativa L.of chicory of Compositae, preparing a lactuca sativa L.of Araliaceae, preparing a lactuca sativa L.of Lactuca sativa L.of Compositae, preparing a lactuca sativa L.of Lactuca, and preparing a lactuca sativa L.of Lactuca, wherein lactuca sativa L.of Lactuca sativa L.of Compositae, is a L.of Lactuca sativa L.of Lactuca, is a L.of Lactuca sativa L.A.A.A.A.A.A.A, is a L.A.of Lactuca, is a L.of Lactuca, is a L.A, is a L.A.A.A.A.A, is a L.A.A, is a L.

Disclosure of Invention

The invention aims to provide a method for preparing lactucin, which takes a cichorium intybus root extract as a raw material, respectively adopts reversed phase C18 preparative chromatography and glucan L H-20 type gel chromatography, separates a component solution rich in lactucin from the cichorium intybus root extract, and obtains high-purity lactucin by a reduced pressure concentration crystallization method.

The preparation method of the lactucin provided by the invention comprises the following steps:

a. pulverizing Cichorium intybus Roxb root, extracting with 70% methanol water solution at a ratio of 1:5 under reflux for 1 hr, repeatedly extracting for 3 times, filtering, mixing, and concentrating under reduced pressure to obtain extract; taking the obtained extract, adding water with twice volume, ultrasonically dispersing into suspension, adding petroleum ether or n-hexane with equal volume for degreasing for 3 times, mixing the obtained water phases, adding ethyl acetate with equal volume for extracting for 3 times, mixing the obtained ethyl acetate layers, concentrating under reduced pressure, and freeze-drying under vacuum to obtain a cichorium intybus root extract;

b. b, ultrasonically dissolving 50-2000mg of the cichorium intybus root extract obtained in the step a in a methanol-water solution with the volume ratio of 50m L being 20-100%, centrifuging to remove precipitates, and taking supernatant for later use;

c. preparing a chromatographic column by taking a reversed phase C18, wherein the specification is that the length is 250mm, the inner diameter is 80mm, the particle size of a filling material is 10 mu m, the mass of the filled filler is 800g, connecting the reversed phase pilot test C18 chromatographic column to a preparative chromatograph, adjusting the mobile phase to be a methanol-0.1% formic acid water mixture, wherein the methanol accounts for 20% -65%, the column temperature is controlled to be minus 10 ℃ -50 ℃, balancing the chromatographic column for 10-30min for later use, controlling the flow rate to be 100-180m L/min, collecting the lactucin peak of the extract solution obtained in the step b, carrying out reduced pressure concentration at the temperature of 50 ℃, and carrying out vacuum freeze drying to obtain 5-170mg of lactucin with the purity of 85%;

d. wet packing a glucan L H-20 type gel chromatographic column with the length of 1.5m and the inner diameter of 7cm by using methanol as a column packing reagent, taking 5-170mg of the lactucin with the purity of 85% obtained in the step c, adding 2-3m L methanol for dissolving, loading the lactucin to the glucan L H-20 type gel chromatographic column, washing the glucan L H-20 type gel chromatographic column by using pure methanol as a mobile phase, collecting effluent components to obtain components with the volume of 5m L in each bottle, collecting 20 bottles, respectively measuring the purity of the lactucin in the obtained 20 bottles of components by using a high performance liquid chromatograph, combining the component solutions with the purity of more than 95%, and concentrating by using a rotary evaporator at the temperature of 50 ℃ until the lactucin is dried;

e. concentrating and drying the solid obtained in the step c, adding methanol to dissolve the solid into clear saturated solution at the temperature of 40-70 ℃, placing the solution in a refrigerator at the temperature of 4 ℃ overnight to obtain colorless transparent crystals, filtering the crystals, washing the crystals with petroleum ether to obtain a lactucin reference substance with the purity of 99%, filtering the crystal mother solution to obtain a crystal mother solution, concentrating the crystal mother solution by using a rotary evaporator at the temperature of 50 ℃ to dry the crystal mother solution, adding methanol to dissolve the crystal mother solution into clear saturated solution at the temperature of 40-70 ℃, placing the crystal mother solution in a refrigerator at the temperature of 4 ℃ overnight to obtain colorless transparent crystals, filtering the crystal mother solution, washing the crystals with petroleum ether, and combining the two batches of crystals to obtain the lactucin reference substance with the purity of 99% of 3-105 mg.

In step b, the content of the extract of the cichorium hirsutum root is 300-1000mg, and the content of methanol in the methanol-water for dissolving the sample of the cichorium hirsutum extract is 50-90%.

In the purification process of the reverse phase C18 preparative chromatography using the filler with the mass of 800g in the step C, the volume ratio of methanol in the mobile phase is 30-50%, and the column temperature is-10-30 ℃.

The crystallization temperature in step e is 60-70 ℃.

The preparation method of lactucin is characterized by that on the basis of the invented patent 201110213474.0 "preparation method of lactucin and lactucin", the lactucin root is pulverized, and the methanol aqueous solution whose concentration is 70% is used, the material-liquid ratio is controlled at 1:5, and the heated reflux extraction is 1H, and the repeated extraction is 3 times, and then the above-mentioned materials are filtered, combined and decompressed and concentrated to obtain extract, and the obtained extract is added with twice volume of water, and ultrasonically dispersed into suspension, and the isometric petroleum ether or n-hexane is added to degrease for 3 times, and the obtained aqueous phases are combined, and the isometric ethyl acetate is added to extract for 3 times, and the obtained ethyl acetate layers are combined, decompressed and concentrated, and vacuum freeze-dried to obtain the extract of lactucin root, then the extract is respectively undergone the processes of reversed phase C18 preparation chromatography and dextran L H-20 type gel chromatography, and the component solution rich in lactucin is separated from the lactucin root extract, and the lactucin can be obtained by means of making lactucin with high purity, and having a certain application value in the standard method for preparing lactucin with high purity.

Detailed Description

The present invention will be described in further detail with reference to the following examples.