CN102617718B - Bran coat antitumor active protein, as well as preparation method and applications thereof - Google Patents
Bran coat antitumor active protein, as well as preparation method and applications thereof Download PDFInfo
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- CN102617718B CN102617718B CN201210084160.XA CN201210084160A CN102617718B CN 102617718 B CN102617718 B CN 102617718B CN 201210084160 A CN201210084160 A CN 201210084160A CN 102617718 B CN102617718 B CN 102617718B
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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
The present invention relates to preparation and the application of vegetable-protein, specifically belong to a kind of cavings anti-tumor active protein and preparation method thereof, and the application of this albumen in preparing antitumor drug and protective foods.
Background technology
Millet, i.e. grain (Setaria italica), annual gramineae platymiscium, originates from China Huanghe valley, and it is global 4/5ths that the annual production of China accounts for, and North China is major production areas, and wherein Shanxi is the main place of production of millet.Cavings is that millet is processed into the byproduct obtaining in polished rice process, is called cavings on technology.
< < Huang di's Canon of Medicine > > thinks that " essence of grain communicating to the spleen, " cavings taste is sweet, and property is flat, and relatively tonifying Qi, has and replenish qi to invigorate the spleen, nourishing blood to tranquillize the mind, the effect of kidney-tonifying and brain tonic.Cavings and the cereal same sex, merit is better than rice with rice, tonify without causing stagnation, warm and not dry, in cavings, contain abundant triglyceride level, lipoprotein, Hi-Z, vitamins B, vitamin-E, glucose, Mierocrystalline cellulose and trace element etc., can discharge the toxin in human body, have brain tonic, kidney tonifying, keep fit, the effect such as anticancer, often eat cavings, can strengthen the immunologic function of human body, illness prevention and fitness.So the deep development of this resource of cavings and effectively utilization will bring very high economy and social value.
Through the literature search of prior art is found, the domestic and international research about cavings at present mainly concentrates on its chemical composition and nutritive value aspect, and has no the report about cavings anti-tumor active protein.Cavings albumen prepared by the present invention has stronger anti-tumor activity, and normal cell is had no side effect.This discovery has potential economic worth for the deep development of cavings and the utilization of effectively rising in value.
Summary of the invention
The object of the present invention is to provide a kind of cavings anti-tumor active protein and preparation method thereof, and the application of this albumen in preparing antitumor drug and protective foods.
A kind of cavings anti-tumor active protein provided by the invention, makes by the method comprising the steps:
The first step: get cavings, after pulverizing, add the protein extract of precooling, soak at 4 ℃ and stir 12-16 hour, get supernatant liquor after centrifugal, be cavings protein crude extract; Filter residue after centrifugal can repeat to extract 1-2 time;
Described protein extract is 20mmol/L Tris-HCl solution, containing 0.85%NaCl, and 1mmol/L PMSF, pH8.0; Described cavings and the weightmeasurement ratio of protein extract (W/V) are 1: 4-6, preferably 1: 5;
Second step: add the acetone precipitation albumen 1-2 hour of-20 ℃ of precoolings of 2-3 times of volume in cavings protein crude extract, centrifugal, collecting precipitation is placed 20-30min at-20 ℃, and acetone is volatilized completely, and lyophilize obtains cavings crude protein;
The 3rd step: by cavings pH8.0, dissolving of 20mmol/L Tris-HCl damping fluid for crude protein, after centrifugal, get supernatant liquor, the ammonium sulfate powder that first adds 30% saturation ratio by ammoniumsulphate soln saturation computation table (0 ℃), fully after stirring and dissolving, standing 30min, centrifugal, collect supernatant liquor, again to the ammonium sulfate powder that adds 85% saturation ratio in supernatant liquor, after abundant stirring and dissolving, standing 30min is centrifugal, collecting precipitation, with pH8.0, the dissolving of 20mmol/L Tris-HCl damping fluid, dialysis, the concentrated antitumor crude protein of cavings that obtains;
The 4th step: by antitumor pH8.0, the dissolving of 20mmol/L Tris-HCl damping fluid for crude protein, cross Q anion-exchange chromatography post, Ultraviolet Detector detects in 280nm wavelength place, collects and penetrates peak;
The 5th step: the above-mentioned liquid that penetrates is crossed to SP-cation seperation column displacement chromatography post, by pH8.0, the 20mmol/LTris-HCl buffer solution elution containing 0.1mol/LNaCl, Ultraviolet Detector detects in 280nm wavelength place, collects elution peak and obtains cavings anti-tumor activity pure protein (FMB).
The 3rd step, the 4th step, the 5th step are all carried out under 0-4 ℃ of condition.
Adopt SDS-PAGE gel electrophoresis technology, by comparing with standard molecular weight, the molecular weight of estimating cavings anti-tumor active protein is about 36kDa.
Through In Vitro Anti intestinal cancer tumor promotion detection display, this cavings albumen can obviously suppress proliferation of colorectal cancer cells and transfer, with this albumen, processes colorectal cancer cells 48 hours, and average lethality rate is about 40%, and to normal cell growth without obvious toxic-side effects.This albumen can be applied in preparing antitumor drug, also can in protective foods, apply.
Compared with prior art, the cavings albumen of the anti-tumor activity that the present invention obtains detects through anti tumor activity in vitro, this albumen has the activity of good Chinese People's Anti-Japanese Military and Political College's colon-cancer cell, proliferation of colorectal cancer cells and migration is had to obvious restraining effect, and normal cell is not had to obvious toxic-side effects.
Accompanying drawing explanation
Fig. 1 cavings anti-tumor active protein SDS-PAGE electrophorogram (gained pure protein sample after SP column purification, applied sample amount is 3 μ g; FMB: target protein; Marker: standard molecular weight)
The impact of Fig. 2 cavings anti-tumor active protein FMB cell growth
Fig. 3 A cell scratch experiment is observed the impact of cavings anti-tumor active protein on cell migration
Fig. 3 B cell scratch experiment is observed the impact (* 4) of cavings anti-tumor active protein on cell migration
Embodiment
The preparation of embodiment 1 cavings anti-tumor active protein
(1) get 30g cavings, pulverize, add protein extract (the 20mmol/L Tris-HCl solution of precooling at 150mL4 ℃, containing 0.85%NaCl, 1mmol/L PMSF, pH8.0), be placed on agitator and stir, in 4 ℃ of refrigerators, soak and stir 12 hours, the centrifugal 30min of 11000rpm, gets supernatant liquor, and filter residue adds the protein extract of 150mL again, the same operation, merges supernatant liquor and is cavings protein crude extract.
(2) acetone of precooling at-20 ℃ of 600mL will slowly be added in the cavings protein crude extract obtaining in (1), limit edged shakes, then put it at-20 ℃ and precipitate 1 hour, the centrifugal 30min of 11000rpm, abandons supernatant, collecting precipitation, precipitation is put into 20min at-20 ℃, acetone is volatilized completely, will precipitate lyophilize, obtain the cavings crude protein of 75mg.
(3) the cavings crude protein (2) being obtained dissolves with pH8.0, the 20mmol/L Tris-HCl damping fluid of 100mL, after 11000rpm is centrifugal, get supernatant liquor, in solution, by amount, slowly add 16.4g ammonium sulfate powder, saturation ratio reaches 30%, place 30min, the centrifugal 30min of 11000rpm, collects supernatant.By slowly adding again the ammonium sulfate solids of 36.2g in supernatant liquor, reach 85% saturation ratio, place 30min, 11000rpm, centrifugal 30min, collecting precipitation.To precipitate with pH8.0, the dissolving of 20mmol/LTris-HCl damping fluid, dialysis (adopting the dialysis tubing of molecular weight cut-off 10kDa), the concentrated cavings anti-tumor activity crude protein that obtains.
(4) after by the cavings anti-tumor activity of (3) acquisition, for crude protein, pH8.0,20mmol/LTris-HCl damping fluid dissolve, carry out Q anion column displacement chromatography, by pH8.0,20mmol/L Tris-HCl buffer solution elution, Ultraviolet Detector detects in 280nm wavelength place, collects and penetrates peak.
(5) the above-mentioned liquid that penetrates is splined on to SP positively charged ion chromatography post, by the pH8.0 that contains 0.1mol/L NaCl, 20mmol/LTris-HCl buffer solution elution, Ultraviolet Detector detects in 280nm wavelength place, collect elution peak, dialysis, concentrated rear cavings anti-tumor activity pure protein (FMB) 4.668mg that concentration is 0.561mg/mL that obtains.
All carry out under 0-4 ℃ of condition step (3), (4), (5).
Embodiment 2: the impact of cavings anti-tumor active protein FMB cell growth
By the DLD1 of logarithmic phase, SW480, SW620, HL-7702 cell, with 1 * 10
496 well culture plates are imported in individual/hole into.37C contains 5% CO
2incubator is hatched after 24h, adds respectively the cavings activated protein 20 μ L of embodiment 1, establishes five multiple holes, and with the pH8.0 of same volume, 20mmol/L Tris-HCl damping fluid in contrast.After hatching 48h, liquid in hole is abandoned in suction.After PBS washing 2 times, add new substratum, every hole adds MTT solution (5mg/mL) 20 μ L, stops cultivating after continuing to hatch 4h.Careful suction abandoned culture supernatant in hole, and every hole adds 150 μ LDMSO, and vibration 10min, fully melts crystallisate.570nm wavelength place on enzyme linked immunological monitor, measures each hole absorbance value.
Gained cavings activated protein FMB of the present invention, Human Large Intestine Carcinoma Cells strain is carried out to above-mentioned anti tumor activity in vitro detection, result shows: when the final concentration of FMB is 0.09 μ g/ μ L, on the inhibiting rate of DLD1 Growth of Cells be about 40%, on SW480 inhibitory rate of cell growth be about 38%, on SW620 inhibitory rate of cell growth be about 38% and on Human normal hepatocyte HL-7702 growth substantially without affecting (as shown in Figure 2).
Embodiment 3: cell scratch experiment is observed the impact of cavings anti-tumor active protein on cell migration
By DLD1 and HL-7702 cell with 5～10 * 10
5individual/hole density is inoculated on 24 orifice plates, and cell attachment also forms after monolayer cell, and by the aseptic liquid-transfering gun rifle of 10 μ L head vertical score, PBS washing, measures cut diameter, and be respectively equipped with experimental group and control group under microscope.Experimental group adds cavings anti-tumor activity pure protein (FMB) the 100 μ L of embodiment 1, and control group adds pH8.0, the 20mmol/L Tris-HCl damping fluid of same volume.Hatch respectively after 24h, 48h, under microscope, measure the distance that cut place cell outwards moves.
Cell scratch experiment result shows: the cavings activated protein FMB that purifying of the present invention obtains, when the final concentration of FMB is 0.09 μ g/ μ L, migration to large bowel cancer DLD1 cell strain has restraining effect, and this restraining effect all reaches extremely significantly (p < 0.01) when albumen is processed 24h, 48h; And to the migration of normal liver cell HL-7702 without obvious effect (p > 0.05), (as shown in Fig. 3 A, Fig. 3 B).
Claims (4)
1. a cavings anti-tumor active protein, is characterized in that, by the method comprising the steps, makes:
The first step: get cavings, after pulverizing, add the protein extract of precooling, soak at 4 ℃ and stir 12-16 hour, get supernatant liquor after centrifugal, be cavings protein crude extract;
Described protein extract is 20mmol/L Tris-HCl solution, containing 0.85%NaCl, and 1mmol/L PMSF, pH8.0; Described cavings and the weightmeasurement ratio of protein extract are 1 ︰ 4-6, and the unit of wherein wt is g, and the unit of volume is mL;
Second step: in cavings protein crude extract, add the acetone of-20 ℃ of precoolings of 2-3 times of volume, protein precipitation 1-2 hour, centrifugal, collecting precipitation is placed at-20 ℃, and acetone is volatilized completely, and lyophilize, obtains cavings crude protein;
The 3rd step: by cavings pH8.0, dissolving of 20mmol/L Tris-HCl damping fluid for crude protein, after centrifugal, get supernatant liquor, ammoniumsulphate soln saturation computation table at pressing 0 ℃, first add ammonium sulfate powder, reach 30% saturation ratio, fully after stirring and dissolving, standing, centrifugal, collect supernatant liquor, in supernatant liquor, add ammonium sulfate powder again, reach 85% saturation ratio, fully after stirring and dissolving, standing, centrifugal, collecting precipitation, with pH8.0, the dissolving of 20mmol/L Tris-HCl damping fluid, dialysis, the concentrated antitumor crude protein of cavings that obtains;
The 4th step: by antitumor pH8.0, the dissolving of 20mmol/L Tris-HCl damping fluid for crude protein, cross Q anion-exchange chromatography post, Ultraviolet Detector detects in 280nm wavelength place, collects and penetrates peak;
The 5th step: the above-mentioned liquid that penetrates is crossed to SP-cation seperation column displacement chromatography post, by pH8.0, the 20mmol/L Tris-HCl buffer solution elution containing 0.1mol/L NaCl, Ultraviolet Detector detects in 280nm wavelength place, collects elution peak and obtains cavings anti-tumor active protein.
2. a kind of cavings anti-tumor active protein as claimed in claim 1, is characterized in that, the weightmeasurement ratio of the cavings described in the first step and protein extract is 1 ︰ 5, and the unit of wherein wt is g, and the unit of volume is mL.
3. the application of a kind of cavings anti-tumor active protein as claimed in claim 1 in preparation Chinese People's Anti-Japanese Military and Political College bowelcancer medicine.
4. the application of a kind of cavings anti-tumor active protein as claimed in claim 1 in preparing protective foods.
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| CN108048418B (en) * | 2018-01-11 | 2021-02-02 | 山西大学 | Bran coat source peroxidase anti-tumor active fragment, and preparation method and application thereof |
| CN110101850B (en) * | 2019-05-07 | 2023-03-10 | 山西大学 | Application of bran coat peroxidase in preparation of product for preventing and treating atherosclerosis |
| CN110590922B (en) * | 2019-10-15 | 2022-04-22 | 南开大学 | Antitumor protein extract in red onion bacteria, and preparation method and application thereof |
| CN111773334A (en) * | 2020-08-07 | 2020-10-16 | 南开大学 | A beverage and food for preventing and/or treating inflammatory bowel disease or colorectal cancer |
| CN113243446B (en) * | 2021-05-20 | 2022-11-08 | 山西大学 | Chaff protein extract and preparation method and application thereof |
| CN114752585B (en) * | 2022-05-20 | 2023-07-18 | 山西大学 | Walnut green seedcase antitumor active protein and preparation method and application thereof |
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