CN102093345B - Method for separating out dehydrogenized turkey corn by applying silicagel column chromatography - Google Patents

Abstract

The invention discloses a method for separating dehydrocorydalline using silica gel column chromatography: (1) take 200-300 mesh silica gel, add chloroform and stir it, pour it into a chromatography column and pack it into a column; The crude ethanol extract was dissolved in a small amount of solvent mixed with chloroform and methanol at a volume ratio of 50:1 to make a sample solution, and the eluents with a volume ratio of chloroform and methanol at 50:1, 40:1, and 30:1 were sequentially used Carry out gradient elution, the flow rate of the eluent is 1.6~2.5ml/min, collect the eluate in equal volumes, combine the R _f value and retention time of the standard dehydrocorydalline and the purity of dehydrocorydaline is greater than 90% The eluent is recovered with a rotary evaporator to obtain the crude product of dehydrocordyrine; (3) the crude product of dehydrocordyline obtained in step (2) is crystallized with methanol or chloroform after depigmentation to obtain dehydrocordyline Pure. The process of the invention is simple, the recovery rate is high, and the dehydrocorydine obtained by separation has high purity.

Description

A method for separating dehydrocordyrine by using silica gel column chromatography

(1) Technical field

The invention relates to a method for separating and obtaining high-purity dehydrocorydalline from the crude extract of Rhizoma radix by using silica gel column chromatography.

(2) Background technology

Corydalis yanhusuo W.T.Wang is the dry tuber of Corydalis yanhusuo (Popaveraceae), which is pungent, bitter and warm in nature, and returns to the liver and spleen meridian. It is mainly produced in Zhejiang, Jiangsu, Anhui, Sichuan and other provinces. Yuanhu is One of the famous "Eight Flavors of Zhejiang". Yuanhu has the effects of promoting blood circulation, promoting Qi and relieving pain. It can be used for chest and hypochondria, epigastric pain, amenorrhea dysmenorrhea, postpartum stasis, tumbling and swelling. Modern studies have shown that the active ingredients of Rhizoma Rhizoma are mainly alkaloids. According to reports, there are more than 40 species of alkaloids in Rhizoma Rhizoma Rhizoma, mainly including dozens of biological species such as corydalis, protopine, and dehydrocorydaline. alkali. Modern pharmacological experiments have proved that tetrahydropalmatine in the alkaloids contained in Yuanhu is the main analgesic and sedative component, and dehydrocorydine can dilate coronary blood vessels, increase coronary blood flow, improve myocardial nutritional blood flow, and strengthen myocardial blood flow. The ability to resist hypoxia and protect myocardial ischemia and necrosis is the active ingredient of Yuanhu preparation "Karling" for the treatment of coronary heart disease and angina pectoris. In addition, dehydrocorydine has also achieved good curative effect in the treatment of ulcer disease.

At present, there are two kinds of separations of dehydrocordyrine reported in the literature: one is the application of standard high-speed countercurrent chromatography, which has a small amount of sample, and the equipment used is relatively expensive, making it difficult to be widely used; the other is the comprehensive use of various methods. Kinds of column chromatography (macroporous adsorption resin, alumina column chromatography and gel column chromatography), this method steps are loaded down with trivial details, and efficiency is low, and through repeated chromatography, the dead adsorption of sample is bigger.

(3) Contents of the invention

The purpose of the present invention is to provide a method for separating high-purity dehydrocorydaline, which has simple process, high separation efficiency and can be used in industrial production.

Technical scheme of the present invention is as follows:

A method for separating dehydrocordline using silica gel column chromatography, said method comprising the following steps:

(1) Take 200-300 mesh silica gel, add chloroform to stir, place in an ultrasonic oscillator and oscillate until there are no bubbles, then pour it into a chromatographic column and pack it into a column to obtain a silica gel column;

(2) Dissolve the ethanol crude extract of Rhizoma Rhododendron with a small amount of chloroform and methanol with a volume ratio of 50:1 to make a test solution, take the test solution as a sample solution, and make the sample solution completely adsorbed On the silica gel, the mass ratio of the ethanol crude extract of Rhizoma chinensis in the sample solution to the silica gel in the silica gel column is 1:30～105, and the eluent with chloroform and methanol volume ratio of 50:1 is used for elution of 150～150 250min, chloroform, methanol volume ratio of 40:1 eluent elution 150 ~ 250min, chloroform, methanol volume ratio of 30:1 eluent eluent until thin layer chromatography tracked and detected no dehydroviolet in the eluent Cordyline component, the flow rate of the eluent is 1.6-2.5ml/min, the eluate is collected in equal volumes, and the collected eluate is detected by thin-layer chromatography and high-performance liquid chromatography, and combined with dehydrocorydalis The eluent with the same _Rf value and retention time of the alkali standard product and the purity of dehydrocordyrine is greater than 90%, and the solvent is recovered by a rotary evaporator to obtain the crude product of dehydrocordyline;

(3) The crude dehydrocordyrine obtained in step (2) is depigmented and then crystallized with methanol or chloroform to obtain pure dehydrocordyline.

The ethanol crude extract of Rhizoma Rhizoma Rhizoma Rhizome is obtained as follows: grind Rhizoma Rhizoma Rhizoma root, reflux extraction with 60-98% ethanol for 1-5 times, combine the extracts of each time, and concentrate the filtrate under reduced pressure to soak after suction filtration. Paste, use acid solution to adjust the pH value to 2~3, let it stand for 10~30 hours, take the supernatant and filter to remove the precipitate; extract the filtrate with chloroform for 1~5 times, collect and combine the chloroform layers, and recover the chloroform by rotary evaporation to obtain Yuan Hu ethanol crude extract.

The acid solution is a hydrochloric acid aqueous solution or a 1-2% sulfuric acid aqueous solution with a mass concentration of 1-2%.

In the step (3), the method for depigmentation of the crude product of dehydrocordyline is one of the following:

(A) Dissolve the pigment with a small amount of absolute ethanol for the crude product of dehydrocordyline, suck out the pigment with a dropper, and crystallize the residue with methanol or chloroform to obtain the pure product of dehydrocordyline;

(B) Dissolve the crude product of dehydrocorydalline in methanol, add a small amount of activated carbon, the mass and dosage of activated carbon is usually calculated as 0.01-0.02g/ml based on the volume of the obtained solution, stir for 45-60min, remove the activated carbon by filtration, and take the filtrate to remove the solvent by rotary evaporation , and the residue was crystallized with methanol or chloroform to obtain pure dehydrocorydine.

The column loading in the step (1) is to add chloroform to the silica gel and stir, place it in an ultrasonic oscillator to vibrate until there are no bubbles, then pour it into the chromatography column to pack the column, and pressurize it with a double ball until the surface of the silica gel does not drop.

The sample loading in the step (2) is to add the sample solution dropwise with a dropper until the sample is completely adsorbed on the silica gel after the chloroform is flush with the silica gel surface.

In the step (2), the ethanol crude extract of Rhizoma Rhizoma Rhizoma is dissolved in a solvent mixed with a small amount of chloroform and methanol at a volume ratio of 50:1, and the small amount refers to the amount that can completely dissolve the crude ethanol extract of Rhizoma Rhizoma Rhizome That is, it can be understood by those skilled in the art.

The detection condition of high performance liquid chromatography in the described step (2) is: ODS C ₁₈ column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Mobile phase: acetonitrile-0.03mol/L sodium dihydrogen phosphate (32: 68, v/v), isocratic elution; flow rate: 0.6ml/min; detection wavelength: 345nm; injection volume: 20μl.

The obtained pure dehydrocordyrine can be further detected by the above-mentioned detection conditions of high performance liquid chromatography, and it is further confirmed that it is dehydrocordyline with a purity greater than 98%.

The beneficial effect of the present invention is that: the dehydrocorydalline separated by the present invention has high purity, simple process and high recovery rate, and plays an important role in the full utilization of Rhizoma Rhizoma Rhododendron.

(4) Specific implementation methods

The present invention will be further described below with specific examples, but the protection scope of the present invention is not limited thereto.

Example 1

Take 1500g of Rhizoma Rhododendron, grind it, and extract it with 22.5L of 70% ethanol under reflux for 30 minutes, extract 3 times in the same way, combine the extracts for 3 times, and concentrate the filtrate under reduced pressure to extract after suction filtration. Adjust the pH value to 3, let it stand for 15 hours, filter with suction, extract the filtrate with 4×400mL chloroform, combine the chloroform layers and recover the chloroform with a rotary evaporator to obtain 10.4g of crude extract of Rhizoma Rhizoma Rhododendron.

Weigh 25g of 200-300 mesh silica gel, add 300mL of chloroform, stir, place in an ultrasonic oscillator to oscillate until there are no bubbles, then pour it into a chromatography column for packing to obtain a silica gel column.

Weigh 239 mg of the crude extract of Rhizoma Rhizoma (the ratio of sample to silica gel is 1:105), and dissolve it with 4 mL of eluent (chloroform-methanol=50:1 (v/v)) to obtain a sample solution.

Use a dropper to drop all the sample solution onto the surface of the silica gel and make it completely adsorbed on the silica gel.

Sequentially eluted with chloroform, methanol volume ratio of 50:1 eluent for 250min, chloroform, methanol volume ratio of 40:1 eluent for 250min, chloroform, methanol volume ratio of 30:1 eluent for elution to Thin-layer chromatography tracking detects that there is no dehydrocordyrine component in the eluent, and the eluent flow rate is 2.5ml/min. Collect the eluent with an Erlenmeyer flask, collect one bottle per 50ml, and collect the eluate obtained Deliquification is detected by thin layer chromatography and high performance liquid chromatography, and the eluent with the same R _f value and retention time as the dehydrocorydaline standard substance and the dehydrocorydaline purity greater than 90% is combined, and the dehydrocorydaline is purified by a rotary evaporator. The solvent is recovered to obtain the crude product of dehydrocorydine.

The crude product of dehydrocorydine was dissolved in 3 mL of absolute ethanol, sucked out with a dropper, and the remaining components were crystallized with 5 mL of methanol to obtain 26 mg of yellow crystals. Hydrocorydine has a purity of 99.6% and a recovery rate of 84.9% (based on the crude extract of Rhizoma Rhizoma Rhododendron).

The detection conditions of HPLC are: ODS C ₁₈ column (4.6×250mm, 5 μm); column temperature: 30°C; mobile phase: acetonitrile-0.03mol/L sodium dihydrogen phosphate (32:68, v/v), Isocratic elution; flow rate: 0.6ml/min; detection wavelength: 345nm; injection volume: 20μl.

Example 2

Weigh 20g of 200-300 mesh silica gel, add 250mL of chloroform, stir, place in an ultrasonic oscillator to oscillate until there are no bubbles, then pour it into a chromatography column for packing to obtain a silica gel column.

Weighed 368 mg of the crude extract of Rhizoma chinensis extracted in Example 1 (the ratio of sample to silica gel was 1:54), and dissolved it with 5 mL of eluent (chloroform-methanol=50:1 (v/v)) to obtain a sample solution.

Use a dropper to drop all the sample solution onto the surface of the silica gel and make it completely adsorbed on the silica gel.

Sequentially eluted with chloroform, methanol volume ratio of 50:1 eluent for 150min, chloroform, methanol volume ratio of 40:1 eluent for 200min, chloroform, methanol volume ratio of 30:1 eluent for elution to Thin-layer chromatography tracking detects that there is no dehydrocordyrine component in the eluent, and the flow rate of the eluent is 2.0ml/min. Collect the eluent with an Erlenmeyer flask, collect one bottle per 50ml, and collect the eluate obtained Deliquification is detected by thin layer chromatography and high performance liquid chromatography, and the eluent with the same R _f value and retention time as the dehydrocorydaline standard substance and the dehydrocorydaline purity greater than 90% is combined, and the dehydrocorydaline is purified by a rotary evaporator. The solvent is recovered to obtain the crude product of dehydrocorydine.

The crude product of dehydrocorydine was dissolved in 40 mL of methanol, 0.4 g of activated carbon was added, stirred for 45 min, the activated carbon was removed by filtration, the filtrate was taken by rotary evaporation to remove the solvent, and the residue was crystallized with 6 mL of methanol to obtain 37 mg of yellow crystals, which were determined by high performance liquid phase detection. It is dehydrocorydalline, with a purity of 99.6% and a recovery rate of 78.4% (based on the crude extract of Rhizoma chinensis).

The detection condition of high performance liquid chromatography is: ODS C _i8 column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Mobile phase: acetonitrile-0.03mol/L sodium dihydrogen phosphate (32:68, v/v), Isocratic elution; flow rate: 0.6ml/min; detection wavelength: 345nm; injection volume: 20μl.

Example 3

Weigh 9 g of 200-300 mesh silica gel, add 200 mL of chloroform, stir, place in an ultrasonic oscillator to vibrate until there are no bubbles, pour it into a chromatography column and pack it into a column to obtain a silica gel column.

Weighed 296mg of the crude extract of Rhizoma radix obtained in Example 1 (the ratio of sample to silica gel was 1:30), and dissolved it with 4mL of eluent (chloroform-methanol=50:1 (v/v)) to obtain a sample solution.

Use a dropper to drop all the sample solution onto the surface of the silica gel and make it completely adsorbed on the silica gel.

Sequentially eluted with chloroform, methanol volume ratio of 50:1 eluent for 150min, chloroform, methanol volume ratio of 40:1 eluent for 150min, chloroform, methanol volume ratio of 30:1 eluent for elution to Thin-layer chromatography tracking detects that there is no dehydrocordyrine component in the eluent, and the flow rate of the eluent is 1.6ml/min. Collect the eluent with an Erlenmeyer flask, collect one bottle per 50ml, and collect the eluate obtained Deliquification is detected by thin-layer chromatography and high-performance liquid chromatography, and the eluent with the same Rf value and retention time as the dehydrocorydaline standard product and the dehydrocorydaline purity is greater than 90% is combined and recovered by a rotary evaporator Solvent to obtain the crude product of dehydrocorydine.

The crude product of dehydrocorydalline was dissolved in 55 mL of methanol, 1.1 g of activated carbon was added, stirred for 60 min, the activated carbon was removed by filtration, the filtrate was taken by rotary evaporation to remove the solvent, and the residue was crystallized with 5 mL of chloroform to obtain 30 mg of yellow crystals, which were determined to be Dehydrocorydine has a purity of 99.4% and a recovery rate of 78.9% (based on the crude extract of Peucedanum rhizome).

The detection conditions of HPLC are: ODS C ₁₈ column (4.6×250mm, 5 μm); column temperature: 30°C; mobile phase: acetonitrile-0.03mol/L sodium dihydrogen phosphate (32:68, v/v), Isocratic elution; flow rate: 0.6ml/min; detection wavelength: 345nm; injection volume: 20μl.

Claims (4)

1. a method of applying silica gel column chromatography to separate dehydrocorydaline, characterized in that said method may further comprise the steps: (1) Take 200-300 mesh silica gel, add chloroform to stir, place in an ultrasonic oscillator and vibrate until there are no air bubbles, then pour it into a chromatography column to install a column to obtain a silica gel column; (2) Dissolve the ethanol crude extract of Yuanhu with a small amount of chloroform and methanol with a volume ratio of 50:1 to make the test solution, take the test solution as the sample solution, and make the sample solution completely adsorbed On the silica gel, the mass ratio of the ethanol crude extract of Rhizoma chinensis in the sample solution to the silica gel in the silica gel column is 1:30～105, followed by 150～105 eluents with chloroform and methanol volume ratio of 50:1 250min, chloroform, methanol volume ratio of 40:1 eluent elution 150~250min, chloroform, methanol volume ratio of 30:1 eluent eluent until thin layer chromatography trace detection to the eluent without dehydroviolet Cordyline component, the flow rate of the eluent is 1.6~2.5ml/min, the eluate is collected in equal volumes, and the collected eluate is detected by thin-layer chromatography and high-performance liquid chromatography, and combined with dehydrocorydalis The eluent with the same _Rf value and retention time of the alkali standard product and the purity of dehydrocordyrine is greater than 90%, and the solvent is recovered by a rotary evaporator to obtain the crude product of dehydrocordyline; The ethanol crude extract of Rhizoma Rhizoma Rhizoma Rhizome is obtained as follows: take Rhizoma Rhizoma Rhizoma Rhizome and crush it, extract 1 to 5 times with 60-98% ethanol under reflux, combine the extracts of each time, and concentrate the filtrate under reduced pressure after suction filtration to immersion. Paste, use acid solution to adjust the pH value to 2~3, let it stand for 10~30 hours, take the supernatant and filter to remove the precipitate; the filtrate is extracted with chloroform for 1~5 times, the combined chloroform layer is collected, and the chloroform is recovered by rotary evaporation to obtain yuan Hu ethanol crude extract; (3) The crude dehydrocordyrine obtained in step (2) is depigmented and then crystallized with methanol or chloroform to obtain pure dehydrocordyline. 2. The method according to claim 1, characterized in that the acid solution is an aqueous hydrochloric acid solution with a mass concentration of 1 to 2% or an aqueous sulfuric acid solution with a mass concentration of 1 to 2%. the 3. The method according to claim 1, characterized in that in the step (3), the method of crystallizing the crude product of dehydrocorydine with methanol or chloroform after depigmentation is one of the following: (A) Dissolve the pigment with a small amount of absolute ethanol for the crude product of dehydrocordyline, suck out the pigment with a dropper, and crystallize the residue with methanol or chloroform to obtain the pure product of dehydrocordyline; (B) Dissolve the crude product of dehydrocorydalline in methanol, add a small amount of activated carbon, stir for 45-60 min, filter to remove the activated carbon, take the filtrate to remove the solvent by rotary evaporation, and crystallize the residue with methanol or chloroform to obtain pure dehydrocorydaline Taste. 4. The method according to claim 1, characterized in that the detection conditions of high performance liquid chromatography in the step (2) are: ODS C ₁₈ column 4.6×250 mm, 5 μm; column temperature: 30°C; mobile phase: Acetonitrile-0.03mol/L sodium dihydrogen phosphate, volume ratio 32:68, isocratic elution; flow rate: 0.6 ml/min; detection wavelength: 345nm; injection volume: 20μl.