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Bran coat antitumor active protein, as well as preparation method and applications thereof

A technology of anti-tumor activity and grain bran, which is applied in the field of preparation and application of plant protein, can solve the problems such as no report of anti-tumor active protein of grain bran, and achieve good anti-colorectal cancer cell activity effect

Active Publication Date: 2012-08-01
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to the literature search of the prior art, it is found that the current domestic and foreign research on rice bran mainly focuses on its chemical composition and nutritional value, and there is no report on the anti-tumor activity protein of rice bran

Method used

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  • Bran coat antitumor active protein, as well as preparation method and applications thereof
  • Bran coat antitumor active protein, as well as preparation method and applications thereof
  • Bran coat antitumor active protein, as well as preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Preparation of gluten bran antitumor active protein

[0022] (1) Take 30g of rice bran, crush it, add 150mL of protein extract (20mmol / L Tris-HCl solution, containing 0.85% NaCl, 1mmol / L PMSF, pH8.0) pre-cooled at 4°C, and stir on the stirrer , soaked and stirred in a refrigerator at 4°C for 12 hours, centrifuged at 11,000 rpm for 30 minutes, took the supernatant, added 150 mL of protein extract to the filter residue, and performed the same operation as above, and combined the supernatant to obtain the crude gluten protein extract.

[0023] (2) Slowly add 600 mL of acetone pre-cooled at -20°C to the crude gluten bran protein extract obtained in (1), shake while adding, then place it at -20°C for precipitation for 1 hour, and centrifuge at 11,000 rpm After 30 minutes, the supernatant was discarded, the precipitate was collected, and the precipitate was placed at -20°C for 20 minutes to completely volatilize the acetone, and the precipitate was freeze-dried t...

Embodiment 2

[0028] Embodiment 2: the effect of gluten bran antitumor active protein FMB on cell growth

[0029] DLD1, SW480, SW620, HL-7702 cells in the logarithmic growth phase were mixed with 1×10 4 per well into a 96-well culture plate. 37C with 5% CO 2 After incubating in the incubator for 24 hours, 20 μL of the gluten bran active protein of Example 1 was added, and five replicate wells were set up, and the same volume of pH 8.0, 20 mmol / L Tris-HCl buffer was used as a control. After 48 hours of incubation, the liquid in the wells was aspirated and discarded. After washing twice with PBS, new medium was added, and 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation was continued for 4 h before the culture was terminated. Carefully aspirate and discard the culture supernatant in the wells, add 150 μL DMSO to each well, and shake for 10 min to fully dissolve the crystals. Measure the light absorbance of each well at a wavelength of 570 nm on an enzyme-linked immun...

Embodiment 3

[0031] Example 3: Observing the effect of the anti-tumor active protein of rice bran on cell migration by cell scratch experiment

[0032] DLD1 and HL-7702 cells were mixed with 5~10×10 5 Cells / well density was seeded on a 24-well plate, after the cells adhered to the wall and formed a monolayer of cells, vertically streaked with a 10 μL sterile pipette tip, washed with PBS, measured the diameter of the scratch under a microscope, and set up experimental groups and the control group. The experimental group was added with 100 μL of the anti-tumor activity pure protein of corn bran (FMB) of Example 1, and the control group was added with the same volume of pH 8.0, 20 mmol / L Tris-HCl buffer. After incubation for 24h and 48h respectively, the distance of the cells moving outward at the scratch was measured under a microscope.

[0033] The results of the cell scratch test show that the purified gluten bran active protein FMB obtained by the present invention can inhibit the migra...

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Abstract

The invention provides a bran coat antitumor active protein, a preparation method of the protein, and applications of the protein in preparing antitumor drugs and health foods. The preparation method of the protein comprises the steps of pulverizing all natural bran coat, adding protein extracting solution, extracting at low temperature after one night, then adding precooled acetone to precipitate protein at low temperature so as to obtain bran coat protein, adding ammonium sulfate powders with different saturation levels into coarse protein extracting solution for fractional precipitation of the protein, enabling the protein precipitated by the ammonium sulfate to permeate through a Q anion exchange chromatographic column, collecting penetrating fluid, enabling the penetrating fluid to permeate through the Q anion exchange chromatographic column again, performing elution with Tris-NaCl eluant with the concentration of 0.1mol / L, and collecting eluting peaks to obtain the bran coat antitumor active protein (FMB). The protein can obviously restrain the proliferation and the metastasis of colorectal cancer cells and can be applied to preparation of the antitumor drugs and the health foods.

Description

technical field [0001] The invention relates to the preparation and application of plant protein, in particular to a grain bran anti-tumor active protein and a preparation method thereof, as well as the application of the protein in the preparation of anti-tumor drugs and health food. Background technique [0002] Millet, that is, millet (Setaria italica), is an annual grass plant that originated in the Yellow River Basin of my country. my country's annual output accounts for four-fifths of the world's total. North China is the main producing area, and Shanxi is the main producing area of ​​millet. Bran is a by-product obtained during millet processing into polished rice. It is called bran in technology. [0003] "Huangdi Neijing" believes that "grain qi flows through the spleen," and grain bran is sweet in taste, flat in nature, and tends to invigorate qi. Bran is of the same nature as grain, has the same function as rice but is better than rice, tonic but not stagnant, wa...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K1/36C07K1/30C07K1/18A61K38/16A61P35/00A23L1/305
Inventor 李卓玉单树花武海丽李宗伟李砧
Owner SHANXI UNIV
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