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A kind of rehmannia glutinosa extract, its preparation method and its application in promoting the proliferation of cik cells in vitro

A technology of extracts and cell bodies, applied in the field of preparation, rehmannia extract, and promotion of CIK cell proliferation in vitro, can solve problems that have not yet been reported in research

Active Publication Date: 2020-09-08
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 cells in vitro with the above four rehmanthins and their extracts as main components.

Method used

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  • A kind of rehmannia glutinosa extract, its preparation method and its application in promoting the proliferation of cik cells in vitro
  • A kind of rehmannia glutinosa extract, its preparation method and its application in promoting the proliferation of cik cells in vitro
  • A kind of rehmannia glutinosa extract, its preparation method and its application in promoting the proliferation of cik cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Effects of Rehmanthin B, Rehmanthin D, Pyrothin A and Pyrothin B on the Proliferation of CIK Cells in Vitro

[0039] 1. Experimental materials

[0040] Rehmanthin B, rehmanthin D, keratin A and keratin B are self-made, and the purity is above 95%.

[0041] 2. Experimental method

[0042] 1. Isolation of single cells

[0043] Use Ficoll-Hypaque density gradient centrifugation to isolate PBMC from the peripheral blood of healthy volunteers, add 4mL PBS to dilute 4mL whole blood (ratio 1:1), take out the diluted blood and slowly add it to a centrifuge tube containing 4mL Ficoll-Hypaque, be careful not to beat Disturb the liquid surface interface, centrifuge at 2000r / min for 20min, and gently draw the mononuclear cells into another centrifuge tube. Add 5 mL of normal saline, blow the cells evenly, and centrifuge to wash twice.

[0044] 2. Culture of CIK cells

[0045] Add CIK initial culture medium to the PBMC suspended in normal saline, and adjust the cell ...

Embodiment 2

[0054] Example 2: Preparation and activity of Rehmannia glutinosa extract with rehmanthin B, rehmanthin D, and pyroxanthin A as main components

[0055] The preparation method comprises the following steps:

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

[0057] Step S2, dissolving the extract with an appropriate amount of water, first extracting with equal volume of petroleum ether for 3 times to remove impurities, then extracting with dichloromethane for 3 times, combining the dichloromethane extracts, concentrating and drying to obtain the dichloromethane extract;

[0058]Step S3, dissolve the dichloromethane extract with 30% ethanol aqueous solution, mix the resin a...

Embodiment 3

[0072] Example 3: Separation and preparation of rehmanthin B, rehmanthin D, and pyrothin A monomers

[0073] The separation preparation method comprises the steps:

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

[0075] Step S2, dissolving the extract with an appropriate amount of water, first extracting with equal volume of petroleum ether for 3 times to remove impurities, then extracting with dichloromethane for 3 times, combining the dichloromethane extracts, concentrating and drying 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 adsor...

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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 in particular relates to a rehmannia glutinosa extract, a preparation method and its application in promoting the proliferation of CIK cells in vitro. Background technique [0002] Rehmanthin B, Rehmanthin D, Pyrothaxanthin A and Pyrorethin B are natural products isolated from Rehmannia glutinosa. The chemical structures are very similar. The chemical information is shown in the table below. [0003] [0004] At present, there are no research reports on the proliferation of CIK cells in vitro with the above four rehmanthins and their extracts as main components. Contents 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] Above-mentioned object realizes through following technical scheme: [0007] The rehmannia glutinosa extract is prepa...

Claims

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Application Information

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