Application of buckwheat hull flavone in inducing differentiation of preadipocytes

A technology of preadipocytes and buckwheat husk flavonoids, which is applied in the field of biomedicine and can solve the problems of low induction and differentiation efficiency of preadipocytes

Active Publication Date: 2019-12-31
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of low efficiency of induction of preadipocyte differentiation, and to provide the application of buckwheat hull flavonoids in inducing preadipocyte differentiation

Method used

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  • Application of buckwheat hull flavone in inducing differentiation of preadipocytes
  • Application of buckwheat hull flavone in inducing differentiation of preadipocytes
  • Application of buckwheat hull flavone in inducing differentiation of preadipocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Buckwheat hull flavonoids (PBHE, PBHE-M 4 ) preparation

[0032] Sweet buckwheat hulls were purchased from Hongyan Buckwheat Rice Factory in Chifeng City, Inner Mongolia; buckwheat hull flavone extract (Buckwheat hullextract, BHE) was extracted with hot water and high-pressure extraction to extract the total flavonoids in buckwheat hulls, specifically: buckwheat hulls, dried and crushed , weigh 500g buckwheat husk powder, add water, carry out hot water high-pressure leaching twice with a solid-liquid ratio of 1:15, each time for 60min, after the leaching solution is concentrated and freeze-dried, purified by D101 macroporous resin, centrifuged and Evaporation under reduced pressure at 75°C followed by vacuum lyophilization to obtain BHE. After washing the unabsorbed compound with distilled water, elution was performed with 70% ethanol at a flow rate of 6 mL / min until no color was observed. The freeze-dried eluate was used for the purification of buckwheat hu...

Embodiment 2

[0033] Example 2 Toxicity experiment of buckwheat husk flavonoids on 3T3-L1 preadipocytes

[0034] MTS method was used to measure the cytotoxicity of the test sample to the cells in order to determine the optimal intervention concentration; 3T3-L1 preadipocytes were treated with 5×10 3 cells / well (100 µL) were inoculated in 96-well plate, at 37°C, 5% CO 2 and saturated humidity conditions for 24 hours; after the cells were confluent, the medium was removed and replaced with buckwheat hull flavones (PBHE, PBHE-M 4) for 48 hours, the concentration was set as: 10, 20, 40, 80, 100 µg / mL; Remove the medium, add 100 µL of medium to each well, add 20 µL of MTS, at 37°C, 5% CO 2 Incubate in an environment for 1-4 hours, use a microplate reader to read the absorbance value at 512nm, and at the same time, measure the absorbance value at 700 nm to remove the background value caused by excessive cell debris;

[0035] The complete medium formula is: 45 mL DMEM+5 mL FBS (fetal bovine seru...

Embodiment 3

[0037] Example 3T3-L1 preadipocytes induced differentiation

[0038] 3T3-L1 preadipocytes were induced to differentiate using the classical "cocktail" method, such as figure 2 . According to the experimental requirements, 3T3-L1 preadipocytes were inoculated into corresponding culture flasks or culture plates at a certain density, and incubated at 37°C in 5% CO 2 Cultivate in an incubator with saturated humidity, culture the complete medium for 2-3 days, wait until the preadipocytes are completely confluent, and continue to contact inhibition for 2 days, replace with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), Differentiation medium with 1 µM dexamethasone (DXMS) and 10 µg / mL insulin (Insulin, INS) culture medium for 2 days (day 0-2), and then replaced with differentiation medium containing 10 µg / mLINS Incubate for another 2 days (2-4 days). On the 4th day, the culture was continued with complete medium containing 10% FBS, and the medium was replaced every 2 days until t...

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Abstract

The invention discloses an application of buckwheat hull flavone in inducing differentiation of preadipocytes. The application comprises the following steps: 1) inoculating and culturing the preadipocytes; after the preadipocytes are completely converged, continuing contact inhibition for 2 days; 2) sucking out the cells, culturing the cells in a differential medium added with buckwheat hull flavone for 2 days, and then culturing the cells in the differential medium added with buckwheat hull flavone for 2 days, wherein the final concentration of buckwheat hull flavone is 10-100 [mu] g/mL; (3)continuously culturing by using a complete culture medium containing 10% of FBS, replacing the culture medium once every two days until 90% of cells show adipose cell phenotypes, and ending the culture. The method is used for inducing the cell differentiation for 6-8 days; the method has the advantages that under the synergistic effect of rosiglitazone, the buckwheat hull flavone can obviously accelerate the cell differentiation process, the differentiation rate of preadipocytes is obviously higher than that of preadipocytes singly added with the buckwheat hull flavone and rosiglitazone, and the induced differentiation time of preadipocytes is shortened.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the application of buckwheat hull flavonoids in inducing differentiation of preadipocytes. Background technique [0002] 3T3-L1 preadipocytes were originally derived from Swiss mouse embryonic tissue. Due to the potential of fibroblasts to induce differentiation into mature adipocytes, this cell line has been widely used to promote the treatment of basic diseases related to obesity, diabetes and related diseases. The study of cellular mechanisms. The differentiation of 3T3-L1 cells is usually induced by a mixture containing the three main components of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX). [0003] It has been found that many factors can affect the differentiation efficiency of 3T3-L1 cells, such as cell line origin, passage number and culture dish number, etc. Mehra et al. found that the differentiation efficiency of 3T3-L1 preadipocytes wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0653C12N2501/999C12N2500/40C12N2501/33C12N2501/39
Inventor 朴春红王海王玥李天竹刘姝妍李云博郭阳王玉华刘俊梅于寒松代伟长
Owner JILIN AGRICULTURAL UNIV
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