CN103087169B - Preparation method of antitumor wheat germ proteins - Google Patents

Abstract

A preparation process of wheat germ anti-tumor protein, comprising: (1) taking fresh wheat germ for low-temperature degreasing and crushing treatment, and then extracting with water as a solvent; (2) removing the mixed extract obtained in step (1) Insoluble matter, and the obtained supernatant was precipitated step by step with ammonium sulfate, and then the precipitate was separated, and then the precipitate was redissolved in water for dialysis treatment, followed by concentration and freeze-drying in turn to obtain wheat Germ water-soluble protein; (3) dissolving the wheat germ water-soluble protein in buffer, and performing ion exchange chromatography and gel filtration chromatography in sequence to obtain the target product. The present invention uses water as the extraction solution, which is natural and healthy, does not cause toxic substances to remain in the obtained product, is beneficial to improving the physiological activity of the obtained product, and has a simple process and low cost. It has significant proliferation inhibitory activity, but has little toxicity to normal cells.

Description

A kind of preparation method of wheat germ anti-tumor protein

technical field

The invention relates to a preparation method of anti-tumor protein, in particular to a preparation method of wheat germ anti-tumor protein, which belongs to the deep processing technology of wheat.

Background technique

Wheat germ is a by-product of flour processing. The production of wheat flour in my country is huge, so the output of wheat germ is also considerable. In addition to high-quality protein, wheat germ also contains vitamin E, magnesium, pantothenic acid, phosphorus, thiamine, etc. It is a rare natural healthy food resource.

Many kinds of anti-tumor active proteins have been found in plants. Foreign researchers have isolated polypeptide components from wheat germ fermentation extracts and proved that they have anti-tumor effects. The physiological activities of wheat germ protein and polypeptide, such as anti-oxidation, anti-aging, and anti-fatigue, have attracted extensive attention from scholars at home and abroad, but there are few reports on its anti-tumor activity. It has been reported that the water-soluble extract of wheat germ can significantly inhibit the growth of S-180 sarcoma, increase the weight of immune organs in tumor-bearing mice, restore the activity of peritoneal macrophages in tumor-bearing mice, and improve the macrophages of mice. The phagocytosis of phagocytic cells, and the water extract has a significant inhibitory effect on the growth of MDA-MB231 cells, but it is not clear which components play a role. Due to the unavoidable solvent residues and unclear active ingredients in fat-soluble extracts, salt-soluble extracts and fermented extracts, it brings certain uncertainty to the development of anti-tumor health food or drugs using wheat germ as raw materials, and it cannot be efficiently Realize the value-added transformation of wheat germ resources efficiently.

Contents of the invention

The purpose of the present invention is mainly to provide a preparation process of wheat germ anti-tumor protein, which uses water as the extract, which is natural and healthy, not only does not cause toxic and harmful substances to be retained in the obtained wheat germ protein, but also helps to improve the quality of the obtained wheat germ protein. The multiple physiological activities of the germ protein make it exhibit significant proliferation inhibitory activity on the growth of human breast cancer cell line MDA-MB231 and low toxicity to normal cells, thereby overcoming the deficiencies in the prior art.

For realizing above-mentioned purpose of the invention, the technical scheme that the present invention adopts is as follows:

A preparation process of wheat germ anti-tumor protein, comprising:

(1) Take fresh wheat germ and carry out low-temperature degreasing and crushing treatment, and then use water as a solvent to carry out extraction operation;

(2) Remove the insoluble matter in the mixed extract obtained in step (1), and carry out fractional precipitation on the obtained supernatant with ammonium sulfate, then separate the precipitate, and then redissolve the precipitate in water for dialysis treatment , followed by concentration and freeze-drying to obtain wheat germ water-soluble protein;

(3) dissolving the wheat germ water-soluble protein in a buffer solution, and performing ion exchange chromatography and gel filtration chromatography in sequence to obtain the target product.

As one of the more preferred embodiments, the step (1) includes: degreasing the wheat germ by means of low-temperature extraction until the residual oil value in the wheat germ meal is below 2.0 wt %;

The low-temperature extraction includes supercritical carbon dioxide extraction, No. 4 solvent pressure extraction or organic solvent low-temperature extraction.

The organic solvent may be selected from but not limited to n-hexane, petroleum ether or acetone.

The low temperature refers to a temperature below 40°C.

As one of the more preferred embodiments, step (1) includes: crushing the defatted wheat germ meal and passing it through a 80-100 mesh sieve to obtain defatted wheat germ powder, and then using a 10:1 (v/w) liquid feed Mix water with defatted wheat germ flour and stir to extract to form a mixed extract.

As one of the more preferred embodiments, step (2) includes: centrifuging the mixed extract obtained in step (1), separating the insoluble matter therein, collecting the supernatant at the same time, and slowly adding ammonium sulfate step by step to the top The precipitation of the precipitate stops in the clear liquid, and then the precipitate is separated by centrifugation, and then the precipitate is redissolved in water, followed by dialysis, concentration and freeze-drying in sequence.

The aforementioned "step-by-step" refers to slowly adding ammonium sulfate to a relatively low saturation state, separating and removing the salted-out protein, and then adding ammonium sulfate to a relatively high saturation state, and then centrifuging to separate the salted-out protein , so as to obtain a relatively pure target protein and remove impurity proteins, which is also a step-by-step salting-out.

Particularly preferably, step (2) also includes: extracting the insoluble matter with water again, then centrifuging again to separate the insoluble matter, and collecting the supernatant again, and then adding ammonium sulfate for step-by-step fractional precipitation, so that The process of step-by-step fractionation precipitation includes:

At room temperature, add ammonium sulfate to the supernatant to 15-20% saturation, precipitate protein, centrifuge, collect the supernatant, add ammonium sulfate to 20-40% saturation, and collect the precipitated protein fraction.

Particularly preferably, the centrifugation in step (2) is performed at a low temperature, and the conditions of the low temperature centrifugation include: the rotation speed is above 8000r/min, the temperature is 0-4°C, and the time is above 20min.

As one of more preferred embodiments, step (3) comprises: described wheat germ water-soluble protein is dissolved in Tris-HCl damping fluid, and pass through DEAE-Sepharose FF ion-exchange chromatography column and Superdex20010/300-GL gel successively Filter the chromatographic column to obtain the target product.

As one of the more preferred embodiments, the conditions of the ion exchange chromatography treatment and gel filtration chromatography treatment described in step (3) include: using a buffer as the eluent, and the flow rate of the eluent is respectively 1.0～2.0mL/min, 0.2～0.5mL/min.

As one of the more preferred embodiments, the preparation process of the wheat germ anti-tumor protein specifically includes:

(1) Take the fresh wheat germ after removing impurities and carry out low-temperature degreasing, then air-dry the defatted wheat germ meal and pass it through a 60-80 mesh sieve to remove the attached fine powder, and then crush it through a 80-100 mesh sieve to obtain defatted wheat germ germ powder;

(2) Mix water and defatted wheat germ flour at a liquid-to-material ratio of 8:1-12:1 (v/w), shake and extract at a speed of 180-220r/min for more than 3 hours, then centrifuge at low temperature, and collect the supernatant liquid, and extract the separated solid matter and water according to the ratio of liquid to material of 4:1 to 6:1 for more than 1 h, then centrifuge again at low temperature, combine the supernatant, and slowly add ammonium sulfate step by step until reaching saturation, and then Stand still for more than 3 hours, centrifuge at low temperature to remove the precipitate, reconstitute with water, put it into a dialysis bag, place it in water below 4°C for dialysis for more than 24 hours, concentrate and freeze-dry to obtain the wheat germ water-soluble protein;

(3) Dissolve the wheat germ water-soluble protein in pH 7.4-8.0, 10-20mmol/LTris-HCl buffer solution, and make the concentration of the formed wheat germ water-soluble protein solution 5-10mg/mL, and then load the sample On the DEAE-Sepharose FF anion-exchange column equilibrated with loading buffer, and pH 7.4-8.0, 10-20mmol/L Tris-HCl buffer containing 0-0.8mol/L NaCl as the eluent, according to 1.0-2.0mL /min flow rate for elution, collect the eluate and dialyze in water below 4°C for more than 24 hours, freeze-dry, and separate the anti-tumor active components;

(4) Dissolving the anti-tumor active component in a pH value of 7.4, containing 50-150mmol/L NaCl, 30-60mmol/L phosphate buffer, and making the concentration of the formed anti-tumor active component solution be 2-4mg/L mL was loaded on the equilibrated superdex20010/300GL gel column, and eluted with buffer at a flow rate of 0.4mL/min to obtain the target product.

As one of the more preferred embodiments, the saturation of ammonium sulfate at 0° C. in step (2) is 20 wt % to 40 wt %.

Compared with the prior art, the present invention has at least the following beneficial effects:

(1) The present invention uses water as a solvent, which is natural and healthy, not only does not cause toxic and harmful substances to be retained in the obtained wheat germ protein, but also helps to improve various physiological activities of the obtained wheat germ protein, and the process of the present invention is simple and low cost. Inexpensive and easy to scale up;

(2) The product prepared by adopting the present invention is white flocculent, single component, and has significant proliferation inhibitory activity to the growth of human breast cancer cell line MDA-MB231, and the IC50 value is 22.33 μ g/mL, and to normal cells Very little toxicity.

(3) The invention provides an effective way for the deep development and utilization of wheat germ.

Description of drawings

Fig. 1 is a process flow diagram for the preparation of a wheat germ anti-tumor protein in a preferred embodiment of the present invention;

Fig. 2 is the DEAE-Sepharose FF chromatogram of WGWSP in a preferred embodiment of the present invention, wherein: WGWSP1 is 0mol/L NaCl elution peak, WGWSP2 is 0.3mol/L NaCl elution peak, WGWSP3 is 0.8mol/L NaCl elution peak;

Fig. 3 is a gel filtration chromatogram of Superdex20010/300GL in a preferred embodiment of the present invention.

Fig. 4 is WGWSP11 high performance liquid phase collection of illustrative plates in a preferred embodiment of the present invention;

Fig. 5 is an SDS-PAGE Coomassie Brilliant Blue staining image of WGWSP11 obtained in a preferred embodiment of the present invention, wherein 1 means adding mercaptoethanol, M means standard protein, and 2 means not adding mercaptoethanol.

Detailed ways

One aspect of the present invention aims to provide a preparation process of wheat germ anti-tumor protein, which may include: using fresh wheat germ as raw material, through low-temperature extraction (such as supercritical carbon dioxide extraction, No. 4 solvent pressure extraction, organic solvent Low-temperature extraction) degreasing, pulverization, shaking water extraction, centrifugation, collecting supernatant, adding ammonium sulfate fractional precipitation, centrifugation, collecting precipitate, redissolving, dialysis, freeze-drying to obtain wheat germ water-soluble protein (WGWSP), WGWSP dissolved Go up DEAE-Sepharose FF ion-exchange chromatography in the buffer solution, separate and obtain the antitumor active component (WGWSP1), (WGWSP1) is dissolved in the buffer solution and go on the Superdex20010/300GL gel filtration chromatography column, obtain the target product (anti-tumor active component (WGWSP1) Tumor Active Component, WGWSP11).

As one of the preferred specific application schemes, referring to Fig. 1-Fig. 5, the preparation process may include:

(1) Preparation of wheat germ water-soluble protein WGWSP: After removing impurities, fresh wheat germ was soaked in n-hexane to degrease, and n-hexane was replaced every 2 hours until the supernatant dripped on the filter paper was air-dried and free of oil marks. Air-dry the degreased wheat germ, sieve to remove the attached fine powder, pulverize and sieve with a swing-type high-speed traditional Chinese medicine grinder to obtain the fine powder, which is defatted wheat germ powder.

(2) Use water as the extraction solvent, the ratio of solid to liquid is 1:10, extract on an air bath oscillator at a speed of 180-220r/min for 3h, then centrifuge at low temperature, extract the residue with half of the water for 1h, centrifuge under the same conditions, and combine To the supernatant, slowly add ammonium sulfate solid to carry out step-by-step precipitation, then centrifuge at low temperature, redissolve the precipitate with water, then put it into a dialysis bag, place it in 4°C water for dialysis for 24 hours, freeze-dry after concentration, and prepare Get WGWSP;

(3) DEAE-SepharoseFF ion exchange chromatography and detection of antitumor activity of each chromatographic component

WGWSP, a freeze-dried sample of wheat germ water-soluble protein, was dissolved in pH 8.0, 20mmol/LTris-HCl buffer solution, the concentration of WGWSP was 5-10mg/mL, passed through a 0.22μm microporous membrane, and loaded on the Liquid-balanced DEAE-Sepharose FF anion-exchange column was eluted with 0, 0.3, 0.8mol/L NaCl pH8.0, 20mmol/LTris-HCl buffer as the eluent, the flow rate was 1.6mL/min, HD- 5 detector detection, the detection wavelength is 280nm, and the automatic collector collects one tube every 5 minutes. The three components WGWSP1, WGWSP2, and WGWSP3 were obtained, which were dialyzed in water at 4°C for 24 hours for desalination, freeze-dried, and the anti-tumor active component WGWSP1 was isolated;

Anti-tumor activity detection: Human breast cancer cell line MDA-MB231 was used as the test cell line, and the anti-tumor activity of WGWSP1, WGWSP2, and WGWSP3 was determined by MTT method. The test shows that WGWSP1 has a strong effect on human breast cancer cell line MDA-MB231. growth inhibitory activity.

(4) Superdex20010/300GL gel filtration chromatography and detection of antitumor activity of each chromatographic component

Dissolve WGWSP1 at 2-4mg/mL in pH7.4, containing 150mmol/L NaCl, 50mmol/L phosphate buffer, pass through a 0.22μm microporous membrane, and load the sample on superdex20010 on the balanced AKTApurifier10 protein purification system /300GL gel column, eluted with buffer, flow rate 0.4mL/min, collected each peak to detect its anti-tumor activity to obtain the active peak WGWSP12.

Anti-tumor activity detection: Human breast cancer cell line MDA-MB231 was used as the test cell line, and the anti-tumor activity of WGWSP11, WGWSP12, WGWSP13, and WGWSP14 was determined by MTT method. The test shows that WGWSP11 has a certain effect on human breast cancer cell line MDA-MB231. Strong anti-proliferation activity. By measuring the anti-proliferation activity of human embryonic kidney cells HEK-293, it was found that the IC ₅₀ of WGWSP11 against normal cells was 191.719 μg/mL, which was far greater than the IC ₅₀ value of MDA-MB231, which was 22.33 μg /mL.

(5) Purity identification of WGWSP11

The purity of WGWSP12 was identified by SEC-HPLC, using Hitachi high performance liquid phase system and Shodex KW-804 protein column. The elution limit of the column is 1,000,000Da, the separation column efficiency is greater than 20,000Da, the mobile phase is 50mmol/L phosphate buffer (pH7.0), the elution rate is 1ml/min, and the eluent is detected at 280nm. The column temperature is room temperature. It is verified that WGWSP12 is a symmetrical peak with a retention time of 11.84min.

(6) Molecular weight distribution of WGWSP12

The molecular weight of WGWSP11 was identified by reducing and non-reducing polyacrylamide gel electrophoresis, which proved that WGWSP12 was a single band, an electrophoretic pure protein, with a molecular weight of about 41kDa.

The technical solution of the present invention will be further described below in conjunction with several preferred embodiments.

Example 1

Add 400mL of water into a 500mL Erlenmeyer flask containing 40g of defatted wheat germ powder, extract on an air bath shaker at a speed of 200r/min for 3h, then centrifuge at 10000r/min, 4°C for 20min to obtain a supernatant, and use 200mL corresponds to solvent extraction for 1h, centrifuge under the same conditions, combine the supernatant, add 63.6g of solid ammonium sulfate to 20% saturation, stir for 40min, let stand for 3h, then centrifuge at 10000r/min, 4°C, 20min, Collect the supernatant, continue to add 72g of solid ammonium sulfate until the saturation is 40%, stir for 40min, let it stand for 3h, then centrifuge at 10000r/min, 4°C for 20min, discard the supernatant to collect the precipitate, add a small amount of water to redissolve the precipitate, Then put it into a dialysis bag, dialyze in water at 4°C for 24 hours, concentrate and freeze-dry to prepare WGWSP. Take 220mg of wheat germ water-soluble protein freeze-dried sample (WGWSP), dissolve in 10mL Tris-HCl buffer, add to DEAE-Sepharose FF ion-exchange column, successively use pH8.0, 20mmol/LTris-HCl buffer, containing 0.3 mol/L NaCl Tris-HCl buffer and 0.8 mol/L NaCl-containing Tris-HCl buffer were used as eluents for elution, the flow rate was 1.6 mL/min, the collected fraction 1 (WGWSP1) was desalted by double distilled water dialysis, frozen dry. 5 mg of WGWSP1 was dissolved in 2 mL of phosphate buffer, added to a Superdex_200 gel filtration column, eluted with phosphate buffer, and fraction 3 was collected, desalted by double-distilled water dialysis, and freeze-dried to prepare WGWSP11.

The wheat germ anti-tumor protein WGWSP11 prepared by this method is white flocculent, with a relative molecular mass of about 41kDa, and has significant proliferation inhibitory activity on the growth of human breast cancer cell line MDA-MB231, with an IC50 value of 22.33 μg/mL. Human embryonic kidney cytotoxicity of normal cells is very small, and the IC50 value of human embryonic kidney cells is 191.72 μg/mL.

Example 2

Add 2000mL water into a 2500mL Erlenmeyer flask containing 200g defatted wheat germ powder, extract on an air bath shaker at a speed of 200r/min for 3h, then centrifuge at 10000r/min, 4°C for 20min to obtain a supernatant, and use 1000mL corresponds to solvent extraction for 1h, centrifuge under the same conditions, combine the supernatant, add 318g of solid ammonium sulfate to 20% saturation, stir for 40 minutes, let stand for 3h, then centrifuge at 10000r/min, 4°C, 20min, Collect the supernatant, continue to add 360g of solid ammonium sulfate until the saturation is 40%, stir for another 40 minutes, let stand for 3h, then centrifuge at 10000r/min, 4°C for 20min, discard the supernatant to collect the precipitate, and add a small amount of water to the precipitate to reconstitute Then put it into a dialysis bag, dialyze in water at 4°C for 24 hours, concentrate and freeze-dry to prepare WGWSP. Take 180mg of wheat germ water-soluble protein freeze-dried sample (WGWSP), dissolve in 5mL Tris-HCl buffer, add to DEAE-Sepharose FF ion-exchange column, use pH8.0, 20mmol/LTris-HCl buffer, containing 0.3 mol/L NaCl Tris-HCl buffer and 0.8 mol/L NaCl-containing Tris-HCl buffer were used as eluents for elution, the flow rate was 1.6 mL/min, the collected fraction 1 (WGWSP1) was desalted by double-distilled water dialysis, and frozen dry. 9 mg of WGWSP1 was dissolved in 3 mL of phosphate buffer, added to a Superdex_200 gel filtration column, eluted with phosphate buffer, fraction 3 was collected, desalted by double-distilled water dialysis, and freeze-dried to prepare WGWSP11.

It should be pointed out that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, any modifications and improvements made within the spirit and principles of the present invention etc., should be included within the protection scope of the present invention.

Claims (2)

1. a preparation method of wheat germ anti-tumor protein, characterized in that, comprising: (1) Degreasing the fresh wheat germ after impurity removal by low-temperature extraction until the residual oil value in the wheat germ meal is below 2.0 wt%, then passing the defatted wheat germ meal through a 60-80 mesh sieve after air-drying, Remove the attached fine powder, and then crush through 80-100 mesh sieves to obtain defatted wheat germ powder, wherein the low-temperature extraction includes supercritical carbon dioxide extraction, No. 4 solvent pressure extraction or organic solvent low-temperature extraction; (2) Mix water and defatted wheat germ flour at a liquid-to-material ratio of 10:1 (v/w), shake and extract at a speed of 180-220r/min for more than 3 hours, then centrifuge at low temperature, collect the supernatant, and The separated solids and water were extracted for more than 1 hour according to the liquid-material ratio of 4:1~6:1, then centrifuged again at low temperature, combined the supernatant, and slowly added ammonium sulfate step by step until reaching saturation, and then stood still for more than 3 hours , centrifuge at low temperature to remove the precipitate, reconstitute with water, put it into a dialysis bag, place it in water below 4°C for dialysis for more than 24 hours, freeze-dry after concentration, and obtain the water-soluble protein of wheat germ, wherein the conditions of the low-temperature centrifugation operation include: The speed is above 8000r/min, the temperature is 0-4°C, and the time is above 20min; (3) Dissolve the wheat germ water-soluble protein in pH 7.4-8.0, 10-20mmol/LTris-HCl buffer solution, and make the concentration of the formed wheat germ water-soluble protein solution 5-10mg/mL, and then load the sample On the DEAE-Sepharose FF anion-exchange column equilibrated with loading buffer, and in sequence with 0mol/L, 0.3mol/L, 0.8mol/LNaCl pH value 7.4～8.0, 10～20mmol/LTris-HCl buffer as The eluent was eluted at a flow rate of 1.0-2.0 mL/min, and the eluate was collected and dialyzed in water below 4°C for more than 24 hours, then freeze-dried, and the anti-tumor active components were separated; (4) Dissolve the anti-tumor active component in a pH value of 7.4, containing 50-150mmol/L NaCl, 30-60mmol/L phosphate buffer, and make the formed anti-tumor active component solution at a concentration of 2-4mg/mL The sample was placed on a balanced superdex20010/300GL gel column, and eluted with the phosphate buffer at a flow rate of 0.4 mL/min to obtain the target product. 2. The preparation method of wheat germ anti-tumor protein according to claim 1, characterized in that the saturation of ammonium sulfate at 0°C in step (2) is 20wt%-40wt%.