CN100336830C - Method for extraction and purification of mustard polysaccharide - Google Patents

Abstract

The present invention discloses an extracting purifying method of mustard polysaccharides. In the method, dried mustard products are taken as raw materials; mustard polysaccharide purified products are obtained through extraction, alcohol precipitation, drying, dialysis and purification; the obtained mustard polysaccharide purified products have water soluble total sugar equal to about 15 % of the dried mustard products, water soluble polysaccharides equal to about 9 % of the dried mustard products, protein contents only equal to 0.2% of the dried mustard products and fat contents only equal to 0.4*10<-3>% of the dried mustard products. The extracting purifying method of mustard polysaccharides of the present invention can save a large quantity of organic solvents, not only has low production cost and protects environment, but also can keep integral structures of mustard polysaccharides and has the advantages of simple technological process, labor saving and time saving.

Description

Extraction and purification method of mustard polysaccharide

technical field

The invention relates to a method for extracting mustard polysaccharide from mustard.

Background technique

Mustard is Brassica juncea, a cruciferous plant. Studies in recent years have found that mustard extracts have antioxidant effects and have a certain effect on preventing diabetes. Mustard also has hypoglycemic and blood lipid-lowering effects, which may be related to mustard polysaccharides. Heart-wrapped mustard is cheap and easy to get, and can be planted in winter slack farmland (late rice land), with high and stable yields. This variety is harvested about 75 days after planting, with an output of about 3,500 kilograms per mu. These characteristics of stuffed mustard are very beneficial to the development of its polysaccharides. There is no research report on polysaccharides of mustard (Brassica juncea) abroad. Hasen Qimuge of the Inner Mongolia Nationalities Normal University in China has conducted preliminary research on mustard polysaccharides, but the separation and purification process is very rough, and no pure mustard polysaccharides have been obtained. There is no description of the mustard species, and there is no follow-up report. as follows:

1. Extraction: Weigh 100g mustard sample and degrease with petroleum ether for 24 hours. The degreased sample is extracted in boiling water for 4 hours, filtered, and the residue is extracted 3 times with distilled water. The three extracts are combined, filtered, and concentrated in a water bath at 80°C to a minimum. volume, add 5 times the volume of absolute ethanol, ethanol analysis for 24 hours, and dry the alcohol analyte to obtain a brown dry product.

2. Purification: Dialyze the brown dry product obtained in step 1 with distilled water for 48 hours, replace the water every 2 hours, then extract with n-butanol-chloroform (1:5) mixture, remove a small amount of protein, and then Hydrogen peroxide decolorizes slowly, and the white mustard polysaccharide is obtained by decompression.

The disadvantages of this mustard polysaccharide extraction and purification method are as follows: 1, use a large amount of petroleum ether, which is costly and pollutes the environment; 2, n-butanol-chloroform is toxic, and will lose polysaccharides, and may cause damage to the structure of mustard polysaccharides; 3. H ₂ O ₂ easily destroys the structure of mustard polysaccharides; 4. Use a large amount of toxic solvents to pollute the environment.

Contents of the invention

The purpose of the present invention is to provide a method for extracting and purifying mustard polysaccharides, which is low in cost, environmentally friendly, and can completely maintain the structure of mustard polysaccharides.

Technical scheme of the present invention is such: the extraction and purification method of mustard polysaccharide is realized by following steps:

(1) Extraction: add distilled water to the dried mustard according to the solid-liquid ratio of 1:20-65 (W/V), extract in constant-temperature hot water at 89.5-90.5°C for 1.8-2.2 hours, filter, and discard the filter residue , to obtain crude extract;

(2) Alcohol analysis: the crude extract obtained in step (1) is concentrated to 1/10 to 1/8 of the original volume at 65 to 75° C., then added with ethanol solution to make the ethanol concentration reach 55%, and the alcohol analysis is 2.5 to 1/8 3.5h, the fat-soluble small molecular substances and pigments are preliminarily removed, and the mustard polysaccharide precipitate is obtained;

(3) drying: washing the mustard polysaccharide precipitate obtained in step (2) with absolute ethanol for 2 to 3 times, then washing with a volatile solvent for 2 to 3 times, and freeze-drying to obtain a crude product;

(4) Dialysis: The crude product obtained in step (3) is fully dissolved in an appropriate amount of distilled water, and the insoluble matter is discarded to obtain a supernatant, which is dialyzed in running water for 45 to 51 hours with a dialysis bag, and then dialyzed in deionized water 22-26 hours, change the water every 7-9 hours to further remove reducing sugars, oligosaccharides, inorganic salts, pigments and other small molecular impurities;

(5) Purification: Concentrate the liquid dialyzed in step (4), add it to a DEAE-Sephadex A-50 column, perform gradient elution with 0-1.5 mol/L NaCl, collect in sections, and collect the liquid Detect with phenol-sulfuric acid method, combine the collected solution of polysaccharide peaks, concentrate, dialyze and vacuum-dry to obtain chromatographically pure mustard polysaccharide.

The solid-liquid ratio of mustard dry product and distilled water in the above-mentioned step (1) is 1: 40 (W/V).

The temperature of the hot water in the constant temperature water bath in the above step (1) is 90°C.

The volatile solvent in the above-mentioned step (3) is acetone.

The volatile solvent in the above step (3) is ether.

After adopting the above technical scheme, the water-soluble total sugar in the obtained mustard polysaccharide pure product can reach about 15% of the mustard dry product, the water-soluble polysaccharide can reach about 9% of the mustard dry product, and the protein content is only 0.2% of the mustard dry product , the fat content is only 0.4×10 ^-3 % of dry mustard. The extraction and purification method of the mustard polysaccharide of the present invention can save a large amount of organic solvents, not only has low production cost and is environmentally friendly, but also the mustard polysaccharide can maintain a relatively complete structure. And the technological process is simple, saving time and effort.

Detailed ways

The extraction and purification method of the mustard polysaccharide of the present invention is realized by the following steps:

(1) Extraction: Weigh an appropriate amount of dry stuffed mustard, add distilled water at a ratio of 1:40 (W/V), add hot water in a constant temperature water tank at 90°C for 2 hours, filter with four layers of gauze, The filter residue was discarded to obtain a crude extract.

(2) Alcohol analysis: Concentrate the crude extract obtained in step (1) to 1/10 of the original volume at 70°C with a rotary evaporator, then add absolute ethanol to make the ethanol concentration reach 55%, and conduct alcohol analysis for 3 hours. Fat-soluble small molecular substances and pigments are removed, and mustard polysaccharide precipitates are obtained.

(3) Drying: Wash the mustard polysaccharide precipitate obtained in step (2) with absolute ethanol for 2 to 3 times, then with acetone or ether for 2 to 3 times, and freeze-dry to obtain a crude product.

(4) Dialysis: the crude product obtained in step (3) is added into distilled water for dissolving at a solid-liquid ratio of 1: 15 (W/V), centrifuged at 6000r.p.m for 15min, discarding insolubles to obtain a supernatant, and using a molecular weight cut-off of The 1000 dialysis bag dialyzes the supernatant in running water for 48 hours, then dialyzes it in deionized water for 24 hours, and changes the water every 8 hours to further remove small molecular impurities such as reducing sugars, oligosaccharides, inorganic salts, and pigments.

(5) Purification: Concentrate the dialyzed liquid in step (4) to 1/2 to 2/3 of the original volume, take 2.5ml and put it on a DEAE-dextran gel A-50 column, and DEAE-dextran gel The size of the gel A-50 column is 2.5×42cm, the height of the column bed is 38cm, gradient elution is carried out with 0-1.5mol/L NaCl, the elution rate is 0.75ml/min, and the collection is divided into parts, each tube collects 3ml, A total of 100 tubes were collected, and the collected solution was detected by the phenol-sulfuric acid method. The collected solution of the polysaccharide peak was combined, concentrated, dialyzed and vacuum-dried to obtain chromatographically pure mustard polysaccharide.

Claims (5)

1, the extracting and purifying method of mustard polysaccharide is characterized in that: realized by the following step:

(1) extract: the solid-liquid ratio by 1: 20～65 (W/V) in the leaf mustard dry product adds distilled water, under 89.5～90.5 ℃ in constant-temperature hot water lixiviate 1.8～2.2h, filter, abandon filter residue, crude extract;

(2) alcohol is analysed: 1/10～1/8 back adding ethanolic soln that the crude extract of step (1) gained is concentrated into original volume under 65～75 ℃, make alcohol concn reach 55%, alcohol is analysed 2.5～3.5h, tentatively removes fat-soluble small-molecule substance and pigment, and obtains the mustard polysaccharide throw out;

(3) drying: with mustard polysaccharide throw out absolute ethanol washing 2～3 times of step (2) gained, with volatile solvent washing 2～3 times, lyophilize gets crude product again;

(4) dialysis: the crude product of step (3) gained is fully dissolving in an amount of distilled water, abandon insolubles, get supernatant liquor, with dialysis tubing with the supernatant liquor 45～51h that in flowing water, dialyses, again with the 22～26h that dialyses in the deionized water, per 7～9h changes water one time, further removes small molecular weight impurities such as reducing sugar, oligosaccharides, inorganic salt, pigment;

(5) purifying: will be through the liquid concentration after step (4) dialysis, last DEAE-dextrane gel A-50 post, NaCl with 0～1.5mol/L carries out gradient elution, fraction collection, collect liquid and detect, merge the collection liquid at polysaccharide peak, concentrate with the phenol sulfuric acid process, dialysis vacuum-drying gets the pure mustard polysaccharide of chromatography.

2, the extracting and purifying method of mustard polysaccharide according to claim 1 is characterized in that: the solid-liquid ratio of leaf mustard dry product and distilled water is 1: 40 (W/V) in the above-mentioned steps (1).

3, the extracting and purifying method of mustard polysaccharide according to claim 1 is characterized in that: the temperature of hot water is 90 ℃ in the Water Tank with Temp.-controlled in the above-mentioned steps (1).

4, the extracting and purifying method of mustard polysaccharide according to claim 1 is characterized in that: the volatile solvent in the above-mentioned steps (3) is an acetone.

5, the extracting and purifying method of mustard polysaccharide according to claim 1 is characterized in that: the volatile solvent in the above-mentioned steps (3) is an ether.