Preparation method and application of tartary buckwheat lectin

Abstract

The invention provides a preparation method of a tartary buckwheat lectin (TBL), and an application of the protein in preparation of an antitumor drug. The preparation method comprises the following steps: crushing tartary buckwheat seeds; adding a protein extracting solution to extract at low temperature, and then adding precipitated ammonium sulfate protein; and carrying out desalination, ion-exchange column chromatography and the like to obtain pure tartary buckwheat lectin protein. By adopting the tartary buckwheat lectin provided by the invention, colorectal cancer cells can be specifically killed, proliferation and metastasis of tumors are inhibited, and the tartary buckwheat lectin can be applied to the antitumor drug.

Description

technical field

[0001] The invention relates to the preparation and application of plant functional protein, in particular to a preparation method of tartary buckwheat lectin and the application of the prepared tartary buckwheat lectin in the preparation of anti-colon cancer drugs. Background technique

[0002] Buckwheat belongs to the dicotyledonous Polygonaceae plant, and there are two cultivated species, namely sweet buckwheat and tartary buckwheat. Buckwheat is widely grown as a traditional crop throughout the world. "Compendium of Materia Medica" records that buckwheat "strengthens the stomach, strengthens energy, refreshes the spirit, sharpens the eyes and ears, refines the five internal organs and dregs", "reduces qi and widens the intestines, grinds stagnant stagnation, reduces heat, swelling and pain" and other functions. In particular, tartary buckwheat is regarded as a wild plant abroad, and it is only cultivated and eaten in the Yunnan-Guizhou Plateau, the Himal...

Examples

Embodiment 1

[0017] Embodiment 1: Preparation of tartary buckwheat lectin

[0018] After dehulling and pulverizing the dried tartary buckwheat seeds, pass through a 40-mesh sieve to collect the powder; weigh 100g of the powder, add 1000mL of 20mM acetate buffer solution with pH 4.5, extract at 4°C for 12 hours, and centrifuge at 8000rpm for 20 minutes Remove the precipitate, filter the supernatant with gauze, then add ammonium sulfate to 80% saturation; stir at 4°C for 4 hours to fully precipitate the protein, centrifuge at 8000rpm for 30min, collect the precipitate, and dissolve it with 30mL PBS buffer solution of pH7.0 Redissolve the pellet; load the dissolved pellet on HiPrep TM 26 / 10 desalting column, remove ammonium sulfate, collect protein peaks to obtain crude buckwheat protein extract; load the crude extract on HiTrap TM The DEAE FF weak anion exchange column was used for separation and purification, and the elution peak of sodium chloride was collected, and the collected elution...

Embodiment 2

[0019] Example 2: Preparation of tartary buckwheat lectin

[0020] After dehulling and pulverizing the dried tartary buckwheat seeds, pass through a 40-mesh sieve to collect the powder; weigh 100g of the powder, add 1000mL of 20mM acetate buffer with pH 4.0, extract at 4°C for 4 hours, and centrifuge at 8000rpm for 20min to remove Precipitate, filter the supernatant with gauze, then add ammonium sulfate to 80% saturation; stir at 4°C for 4 hours to fully precipitate the protein, centrifuge at 8000rpm for 30min, collect the precipitate, and dissolve the precipitate with 30mL of PBS buffer solution with pH7.0 Redissolve; load dissolved pellet on HiPrep TM 26 / 10 desalting column, remove ammonium sulfate, collect protein peaks to obtain crude buckwheat protein extract; load the crude extract on HiTrap TM The DEAE FF weak anion exchange column was used for separation and purification, and the elution peak of sodium chloride was collected, and the collected elution peak protein sa...

Embodiment 3

[0021] Example 3: Preparation of tartary buckwheat lectin

[0022] After dehulling and pulverizing the dried tartary buckwheat seeds, pass through a 40-mesh sieve to collect the powder; weigh 100g of the powder, add 1500mL of 20mM acetate buffer solution with pH 4.5, extract at 4°C for 12 hours, and centrifuge at 8000rpm for 20min to remove Precipitate, filter the supernatant with gauze, then add ammonium sulfate to 80% saturation; stir at 4°C for 4 hours to fully precipitate the protein, centrifuge at 8000rpm for 30min, collect the precipitate, and dissolve the precipitate with 30mL of PBS buffer solution with pH7.0 Redissolve; load dissolved pellet on HiPrep TM 26 / 10 desalting column, remove ammonium sulfate, collect protein peaks to obtain crude buckwheat protein extract; load the crude extract on HiTrap TM The DEAE FF weak anion exchange column was used for separation and purification, and the elution peak of sodium chloride was collected, and the collected elution peak ...

technical field

[0001] The invention relates to the preparation and application of plant functional protein, in particular to a preparation method of tartary buckwheat lectin and the application of the prepared tartary buckwheat lectin in the preparation of anti-colon cancer drugs. Background technique

[0002] Buckwheat belongs to the dicotyledonous Polygonaceae plant, and there are two cultivated species, namely sweet buckwheat and tartary buckwheat. Buckwheat is widely grown as a traditional crop throughout the world. "Compendium of Materia Medica" records that buckwheat "strengthens the stomach, strengthens energy, refreshes the spirit, sharpens the eyes and ears, refines the five internal organs and dregs", "reduces qi and widens the intestines, grinds stagnant stagnation, reduces heat, swelling and pain" and other functions. In particular, tartary buckwheat is regarded as a wild plant abroad, and it is only cultivated and eaten in the Yunnan-Guizhou Plateau, the Himal...

Examples

Embodiment 1

[0017] Embodiment 1: Preparation of tartary buckwheat lectin

[0018] After dehulling and pulverizing the dried tartary buckwheat seeds, pass through a 40-mesh sieve to collect the powder; weigh 100g of the powder, add 1000mL of 20mM acetate buffer solution with pH 4.5, extract at 4°C for 12 hours, and centrifuge at 8000rpm for 20 minutes Remove the precipitate, filter the supernatant with gauze, then add ammonium sulfate to 80% saturation; stir at 4°C for 4 hours to fully precipitate the protein, centrifuge at 8000rpm for 30min, collect the precipitate, and dissolve it with 30mL PBS buffer solution of pH7.0 Redissolve the pellet; load the dissolved pellet on HiPrep TM 26 / 10 desalting column, remove ammonium sulfate, collect protein peaks to obtain crude buckwheat protein extract; load the crude extract on HiTrap TM The DEAE FF weak anion exchange column was used for separation and purification, and the elution peak of sodium chloride was collected, and the collected elution...

Embodiment 2

[0019] Example 2: Preparation of tartary buckwheat lectin

[0020] After dehulling and pulverizing the dried tartary buckwheat seeds, pass through a 40-mesh sieve to collect the powder; weigh 100g of the powder, add 1000mL of 20mM acetate buffer with pH 4.0, extract at 4°C for 4 hours, and centrifuge at 8000rpm for 20min to remove Precipitate, filter the supernatant with gauze, then add ammonium sulfate to 80% saturation; stir at 4°C for 4 hours to fully precipitate the protein, centrifuge at 8000rpm for 30min, collect the precipitate, and dissolve the precipitate with 30mL of PBS buffer solution with pH7.0 Redissolve; load dissolved pellet on HiPrep TM 26 / 10 desalting column, remove ammonium sulfate, collect protein peaks to obtain crude buckwheat protein extract; load the crude extract on HiTrap TM The DEAE FF weak anion exchange column was used for separation and purification, and the elution peak of sodium chloride was collected, and the collected elution peak protein sa...

Embodiment 3

[0021] Example 3: Preparation of tartary buckwheat lectin

[0022] After dehulling and pulverizing the dried tartary buckwheat seeds, pass through a 40-mesh sieve to collect the powder; weigh 100g of the powder, add 1500mL of 20mM acetate buffer solution with pH 4.5, extract at 4°C for 12 hours, and centrifuge at 8000rpm for 20min to remove Precipitate, filter the supernatant with gauze, then add ammonium sulfate to 80% saturation; stir at 4°C for 4 hours to fully precipitate the protein, centrifuge at 8000rpm for 30min, collect the precipitate, and dissolve the precipitate with 30mL of PBS buffer solution with pH7.0 Redissolve; load dissolved pellet on HiPrep TM 26 / 10 desalting column, remove ammonium sulfate, collect protein peaks to obtain crude buckwheat protein extract; load the crude extract on HiTrap TM The DEAE FF weak anion exchange column was used for separation and purification, and the elution peak of sodium chloride was collected, and the collected elution peak ...

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