阅读说明：本技术 一种柱色谱-高速逆流色谱法从羊脆木根皮中纯化羊脆木素a的方法 (Method for purifying sheep crispin A from sheep crispin root bark by column chromatography-high-speed countercurrent chromatography ) 是由 周天北 于 2019-10-22 设计创作，主要内容包括：本发明公开了一种柱色谱-高速逆流色谱法从羊脆木根皮中纯化羊脆木素A的方法,包括如下步骤：步骤A、取适量羊脆木根皮粉末,用乙醇水溶液热回流提取,提取液过滤,浓缩至无醇味,乙酸乙酯等体积萃取,乙酸乙酯部分减压浓缩得乙酸乙酯萃取物；步骤B、取乙酸乙酯萃取物加适量乙醇水溶液溶解,脱脂棉过滤,上样于D101大孔吸附树脂柱,先用10倍柱体积的体积百分浓度为30％的乙醇水溶液洗脱除杂,再用体积百分浓度为65％的乙醇水溶液洗脱,收集第6个柱体积的洗脱液,减压回收乙醇后冷冻干燥得到羊脆木素A富集物；步骤C、高速逆流色谱纯化羊脆木素A。本发明提供了的方法不依赖于硅胶柱层析和凝胶柱层析,易于操作和工业化应用。(The invention discloses a method for purifying sheep crispin A from sheep crispin root bark by using column chromatography-high-speed countercurrent chromatography, which comprises the following steps: step A, taking a proper amount of wood fern root bark powder, performing hot reflux extraction by using an ethanol water solution, filtering an extracting solution, concentrating until no alcohol smell exists, performing isovolumetric extraction on ethyl acetate, and concentrating the ethyl acetate part under reduced pressure to obtain an ethyl acetate extract; b, adding a proper amount of ethanol water solution into the ethyl acetate extract to dissolve, filtering absorbent cotton, loading the mixture into a D101 macroporous adsorption resin column, eluting by using 10 times of column volume of 30% ethanol water solution for removing impurities, eluting by using 65% ethanol water solution, collecting 6 th column volume of eluent, recovering ethanol under reduced pressure, and freeze-drying to obtain a sheep brittle wood element A enrichment; and step C, purifying the sheep brittle lignin A by high-speed counter-current chromatography. The method provided by the invention does not depend on silica gel column chromatography and gel column chromatography, and is easy to operate and industrially apply.)

1. A method for purifying sheep crispin A from sheep crispin root bark by column chromatography-high-speed counter-current chromatography is characterized by comprising the following steps:

step A, Total extract extraction and Ethyl acetate extraction

Taking a proper amount of the sheep brittle wood root bark powder, performing hot reflux extraction by using an ethanol water solution, filtering an extracting solution, concentrating until no alcohol smell exists, performing isovolumetric extraction on ethyl acetate, and concentrating the ethyl acetate part under reduced pressure to obtain an ethyl acetate extract;

step B, enriching the sheep brittle lignin A by column chromatography

Dissolving the ethyl acetate extract with a proper amount of ethanol water solution, filtering absorbent cotton, loading the mixture onto a D101 macroporous adsorption resin column (the height of a column bed is 50cm, the diameter of the column bed is 10cm), eluting with 10 times of the column volume of 30% ethanol water solution for removing impurities, eluting with 65% ethanol water solution, collecting the 6 th column volume of eluent, recovering ethanol under reduced pressure, and freeze-drying to obtain the sheep crispin A concentrate;

step C, purifying the sheep brittle lignin A by high-speed counter-current chromatography

Taking dichloromethane, ethyl acetate, ethanol, water (7:5:6:5, V/V) as a solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase, and taking the upper phase and the lower phase with equal volume as a sample dissolving solvent; setting the temperature of the water bath to be 30 ℃, pumping the stationary phase into the chromatographic column after the temperature is stable, opening the main machine, adjusting the rotating speed to be 850r/min, adjusting the eluting mode to be reverse connection and forward rotation, pumping the mobile phase at the flow rate of 3mL/min after the rotating speed is stable, and continuously pumping a proper amount of mobile phase to ensure that the system is fully balanced when the mobile phase flows out from the tail end of the chromatographic column; preparing the sheep brittle lignin A concentrate into a sample solution by using a sample dissolving solvent, injecting a proper amount of the sample solution into a pipeline from a sample injection ring, starting an acquisition button of a chromatographic workstation, detecting the wavelength of 272nm, collecting a chromatographic peak corresponding to the sheep brittle lignin A according to a chromatogram, concentrating and drying.

2. The method of claim 1, wherein: in the step A, the wood crispus root and bark powder is extracted by hot reflux with an ethanol water solution with the volume percentage concentration of 70-80%.

3. The method of claim 2, wherein: the ratio of material to liquid in the hot reflux extraction is 1: 5.

4. The method of claim 2, wherein: extracting under reflux for 2 hr for 3 times.

5. The method of claim 1, wherein: in step A, ethyl acetate was extracted 3 times with equal volume.

6. The method of claim 1, wherein: and in the step B, adding a proper amount of ethanol water solution with the volume percentage concentration of 15% into the ethyl acetate extract for dissolving.

7. The method of claim 1, wherein: in step C, the stationary phase was pumped through the column at a flow rate of 30 mL/min.

8. The method of claim 1, wherein: and C, continuously pumping the mobile phase for 20min to ensure that the system is fully balanced when the mobile phase flows out from the tail end of the chromatographic column.

9. The method of claim 1, wherein: and step C, preparing the sheep crispin A enrichment substance into a sample solution with the concentration of 10mg/mL by using a sample dissolving solvent, and injecting 10mL of the sample solution into a pipeline from a sample injection ring.

Technical Field

The invention belongs to the field of chemistry, relates to a preparation method of a natural compound, and particularly relates to a method for purifying sheep crispin A from sheep crispin root bark by using column chromatography-high-speed counter-current chromatography.

Background

Pittosporium kerrii Craib, a plant of the genus Pittosporum, has been used as folk herb for thousands of years. It has been reported that various chemical components are sequentially separated from extracts of roots, stems, skins and other parts of the wood of sheep crispa, and triterpenes and compounds such as glycosides, sesquiterpenes, carotenoids, sterols and the like are mainly contained according to the structure types. The chemical components have various biological activities, such as pharmacological activities of resisting tumor, inflammation and pain, resisting virus, resisting microorganism, etc.

The sheep brittle lignin A is an isobenzofuranlactone compound (chemical structure is shown in the specification) separated from sheep brittle wood root bark, and has cytotoxic activity to various tumor cells, and the IC50 values of NB4, SH-SY5Y, PC3, A549 and MCF-7 cells are 3.6, 5.2, 8.8, 5.7 and 6.0 mu mol/L respectively (document: 1 new isobenzofuranlactone compound in sheep brittle wood root bark and its cytotoxic activity, Chinese herbal medicine, 2016 year and 12 months at 23 rd period of volume 47).

At present, the preparation method of the sheep crisp lignin A has less research.

Disclosure of Invention

The invention aims to provide a method for purifying sheep crispin A from sheep crispin root bark by using column chromatography-high-speed countercurrent chromatography.

The purpose of the invention is realized by the following scheme:

a method for purifying sheep crispin A from sheep crispin root bark by column chromatography-high speed countercurrent chromatography comprises the following steps:

step A, Total extract extraction and Ethyl acetate extraction

Taking a proper amount of the sheep brittle wood root bark powder, performing hot reflux extraction by using an ethanol water solution, filtering an extracting solution, concentrating until no alcohol smell exists, performing isovolumetric extraction on ethyl acetate, and concentrating the ethyl acetate part under reduced pressure to obtain an ethyl acetate extract;

step B, enriching the sheep brittle lignin A by column chromatography

Dissolving the ethyl acetate extract with a proper amount of ethanol water solution, filtering absorbent cotton, loading the mixture onto a D101 macroporous adsorption resin column (the height of a column bed is 50cm, the diameter of the column bed is 10cm), eluting with 10 times of the column volume of 30% ethanol water solution for removing impurities, eluting with 65% ethanol water solution, collecting the 6 th column volume of eluent, recovering ethanol under reduced pressure, and freeze-drying to obtain the sheep crispin A concentrate;

step C, purifying the sheep brittle lignin A by high-speed counter-current chromatography

Taking dichloromethane, ethyl acetate, ethanol, water (7:5:6:5, V/V) as a solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase, and taking the upper phase and the lower phase with equal volume as a sample dissolving solvent; setting the temperature of the water bath to be 30 ℃, pumping the stationary phase into the chromatographic column after the temperature is stable, opening the main machine, adjusting the rotating speed to be 850r/min, adjusting the eluting mode to be reverse connection and forward rotation, pumping the mobile phase at the flow rate of 3mL/min after the rotating speed is stable, and continuously pumping a proper amount of mobile phase to ensure that the system is fully balanced when the mobile phase flows out from the tail end of the chromatographic column; preparing the sheep brittle lignin A concentrate into a sample solution by using a sample dissolving solvent, injecting a proper amount of the sample solution into a pipeline from a sample injection ring, starting an acquisition button of a chromatographic workstation, detecting the wavelength of 272nm, collecting a chromatographic peak corresponding to the sheep brittle lignin A according to a chromatogram, concentrating and drying.

Preferably, in the step a, the wood crispness root bark powder is extracted by hot reflux with an ethanol aqueous solution with a volume percentage concentration of 70-80%.

Preferably, the feed-to-liquid ratio of the hot reflux extraction is 1: 5.

Preferably, the extraction is carried out 3 times with hot reflux for 2h each time.

Preferably, in step a, ethyl acetate is extracted 3 times in equal volumes.

Preferably, in step B, the ethyl acetate extract is dissolved by adding a proper amount of 15% ethanol aqueous solution by volume.

Preferably, in step C, the stationary phase is pumped through the column at a flow rate of 30 mL/min.

Preferably, in step C, when the mobile phase flows out from the tail end of the chromatographic column, the mobile phase is continuously pumped for 20min to fully balance the system.

Preferably, in the step C, the aristolochin a concentrate is prepared into a sample solution with a concentration of 10mg/mL by using a sample dissolving solvent, and 10mL of the sample solution is injected into the pipeline from the sample injection ring.

Has the advantages that:

the invention provides a method for purifying sheep brittle lignin A from sheep brittle wood root bark by using column chromatography-high-speed counter-current chromatography, which does not depend on silica gel column chromatography and is easy to operate and industrially apply.

Drawings

FIG. 1 is a comparison of HPLC chromatograms of a sheep brittle lignin A enriched material and a sheep brittle lignin A reference substance;

FIG. 2 is a high-speed countercurrent chromatographic separation chromatogram;

FIG. 3 is a comparison of HPLC chromatograms of high-speed countercurrent chromatography purified product and a control of ovine brittle lignin A.

Detailed Description