Rehmannia extract, preparation method and application in promoting CIK cell in-vitro proliferation

A technology of extract and rehmannia, applied in the field of medicine, can solve problems that have not yet been reported in research

Active Publication Date: 2018-06-22
东营凤起生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are no research reports on the proliferation of CIK cell

Method used

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  • Rehmannia extract, preparation method and application in promoting CIK cell in-vitro proliferation
  • Rehmannia extract, preparation method and application in promoting CIK cell in-vitro proliferation
  • Rehmannia extract, preparation method and application in promoting CIK cell in-vitro proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Effects of Rehmanniae B, Rehmanniae D, Pyrogoxin A and Pyrogoxin B on the proliferation of CIK cells in vitro

[0039] 1. Experimental materials

[0040] Rehmannia glutinin B, rehmannia vin D, pyridoxine A and pyridoxine B are self-made with a purity of more than 95%.

[0041] 2. Experimental method

[0042] 1. Isolate a single cell

[0043] PBMC was separated from the peripheral blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation, 4mL whole blood was diluted with 4mL PBS (ratio 1:1), and the diluted blood was taken out and slowly added to a centrifuge tube containing 4mL Ficoll-Hypaque, taking care not to beat Disturb the liquid surface interface, centrifuge at 2000r / min for 20min, gently pipette mononuclear cells into another centrifuge tube. Add 5 mL of physiological saline, gently blow the cells uniformly, and wash twice by centrifugation.

[0044] 2. Cultivation of CIK cells

[0045] Add CIK initial culture medium to PBMC suspended in sal...

Embodiment 2

[0054] Example 2: Preparation and activity of rehmannia glutinosa extract with rehmannia glutinin B, rehmannia glutinin D, and pyroglutinin A as main components

[0055] The preparation method includes the following steps:

[0056] Step S1: Wash the fresh Rehmannia glutinosa slices and dry them at 55°C to obtain the fresh Rehmannia glutinosa slices; extract the fresh Rehmannia glutinosa slices with a 95% ethanol aqueous solution under reflux for 3 times, each for 2 hours, with a solid-to-liquid ratio of 1:20, combine the extracts, Extract under reduced pressure and concentrate;

[0057] Step S2, dissolve the extract with an appropriate amount of water, first extract 3 times with petroleum ether equal volume to remove impurities, then extract 3 times with dichloromethane, combine the dichloromethane extracts, concentrate and dry to obtain the dichloromethane extract;

[0058] Step S3, dissolve the dichloromethane extract with 30% ethanol aqueous solution, mix the resin and load it on t...

Embodiment 3

[0072] Example 3: Separation and preparation of monomers of Rehmanniae B, Rehmanniae D and Pyrogoxin A

[0073] The separation preparation method includes the following steps:

[0074] Step S1: Wash the fresh Rehmannia glutinosa slices and dry them at 55°C to obtain the fresh Rehmannia glutinosa slices; extract the fresh Rehmannia glutinosa slices with a 95% ethanol aqueous solution under reflux for 3 times, each for 2 hours, with a solid-to-liquid ratio of 1:20, combine the extracts, Extract under reduced pressure and concentrate;

[0075] Step S2, dissolve the extract with an appropriate amount of water, first extract 3 times with petroleum ether equal volume to remove impurities, then extract 3 times with dichloromethane, combine the dichloromethane extracts, concentrate and dry to obtain the dichloromethane extract;

[0076] Step S3, dissolve the dichloromethane extract with 30% ethanol aqueous solution, mix the resin and load it on the XDA-1B macroporous adsorption resin column, ...

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Abstract

The invention discloses a rehmannia extract, a preparation method and application in promoting CIK cell in-vitro proliferation. The total content of rehmannin B, rehmannin D and jioglutin A in the extract is not lower than 90%. The preparation method includes the steps that S1, fresh rehmannia slices are subjected to heat reflux extraction by using an ethanol aqueous solution and subjected to vacuum concentration to obtain an extract; S2, the extract is dissolved with water, extracted with petroleum ether to remove impurities and then extracted with dichloromethane, and dichloromethane extraction solutions are combined, concentrated and dried to obtain a dichloromethane extract; S3, the dichloromethane extract is loaded on an XDA-1B macroporous adsorption resin column, eluted with 30% ethanol to remove impurities and then eluted with 75% ethanol, and 8-10 BV of eluent is collected, concentrated and dried to obtain a crude product of therehmannia extract; S4, the crude product of therehmannia extract is loaded to a normal-phase silica column and eluted with a dichloromethane/methanol/isopropanol mixed solvent in the volume ratio of 10:1:0.2, and 6-7 BV eluent is collected, concentrated and dried. The extract can promote CIK cell in-vitro proliferation.

Description

Technical field [0001] The invention belongs to the field of medicine, and specifically relates to a rehmannia glutinosa extract, a preparation method and application in promoting the proliferation of CIK cells in vitro. Background technique [0002] Rehmanniae B, Rehmanniae D, Pyroghine A and Pyroghine B are natural products separated from Rehmannia glutinosa, with very similar chemical structures. The chemical information is shown in the table below. [0003] [0004] At present, there is no research report on the proliferation of CIK cells in vitro by the above-mentioned four kinds of retinoids and extracts with the four kinds of retinoids as main components. Summary of the invention [0005] The purpose of the present invention is to provide a Rehmannia glutinosa extract, a preparation method and its application in promoting the proliferation of CIK cells in vitro. [0006] The above objectives are achieved through the following technical solutions: [0007] A rehmannia glutinosa ...

Claims

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Application Information

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IPC IPC(8): A61K36/804A61P37/02
CPCA61K36/804A61K2236/15A61K2236/17A61K2236/333A61K2236/39A61K2236/55
Inventor 马海周
Owner 东营凤起生物科技发展有限公司
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