CN102898341A - Extracting and purifying method of high-purity sulforaphane - Google Patents

Abstract

The invention discloses a method for extracting high-purity sulforaphane from broccoli seeds; and the method comprises the following steps of: (1) after crushing dry broccoli seeds, adding water to the dry broccoli seeds to hydrolyze the broccoli seeds, so as to obtain a solid-liquid mixture; (2) adding anhydrous ethanol to the mixture; ultrasonically extracting the mixture; and carrying out suction filtration or double-layer gauze filtering on the mixture; (3) concentrating and evaporating ethanol from a filter liquor in a vacuum state; (4) carrying out chromatography on a material liquor by using a macroporous resin column and eluting the material liquor by using an ethanol solution in a gradient way; (5) collecting an eluting solution containing sulforaphane; carrying out chromatography on the eluting solution by using an alumina column; and concentrating a sulforaphane chromatography solution to form an extract so as to obtain a crude sulforaphane product; (6) adding water to the obtained crude product so as to dissolve the crude product; and extracting the dissolved crude product by using a polar solvent; and (7) collecting an organic layer; and concentrating the organic layer in a vacuum state so as to obtain an oily extract, so that the high-purity sulforaphane is obtained. As the high-purity sulforaphane can be directly extracted from the broccoli seeds and broccoli related materials, the method for extracting the high-purity sulforaphane from the broccoli seeds, disclosed by the invention, has the advantages of simple process, stable product, simple equipment and safety in production and is suitable for enterprises for industrially extracting the sulforaphane.

Description

A method for extracting and purifying high-purity sulforaphane

technical field

The invention relates to a method for extracting and purifying active ingredients in natural products, in particular to a method for extracting high-purity sulforaphane from broccoli seeds.

Background technique

According to the statistics of the World Health Organization, the number of deaths due to cancer in the world is as high as 7 million every year. But so far, people have not found a way to conquer cancer. Therefore, studying and controlling tumors is a major topic in the field of life sciences today. Epidemiological and pharmacological studies have shown that where the incidence of cancer is low, the proportion of vegetables and fruits in people's diet is relatively large. The reason is that there are one or more active ingredients in vegetables and fruits that prevent or slow down the development of cancer, such as lycopene in tomatoes, soy isoflavones in soybean products, tannic acid in berries, and cruciferous fruits and vegetables The sulforaphane and indole substances in it have been proved to have good anti-cancer and anti-cancer effects. Among the many vegetables containing anti-cancer active ingredients, cruciferous (cabbage, broccoli, kohlrabi, broccoli, kale, white radish and cabbage, etc.) Under the action of myrosinase, it can be degraded into isothiocyanate anticancer substances. Sulforaphane is one of the isothiocyanates, and it is also the strongest anti-cancer ingredient found in vegetables so far.

Broccoli, also known as green cauliflower, broccoli, etc., is a variety of Brassica Brassica Brassica species that uses green curds as products. Studies have shown that broccoli is rich in sulforaphane, which can effectively fight breast cancer and colorectal cancer. Healthy people often eat broccoli can also play a role in preventing cancer. Therefore, broccoli is known as a "rookie in cancer prevention".

At present, there are some patent applications for extracting sulforaphane from broccoli, such as "A Method for Extracting Sulforaphane (CN101143842)", "A Broccoli Seed Extract and Its Preparation Method (CN101229211)", " A method for preparing sulforaphane (CN1982294)" and "A method for extracting sulforaphane (CN101544995)", both disclose the extraction and purification methods of the active ingredient sulforaphane in broccoli seeds. The problems in these methods are: silica gel column chromatography is used in the purification process, and then C ₁₈ preparative chromatography or gel column is used to purify sulforaphane, and toxic organic solvent is used for elution in the silica gel column developer, and there are still problems in the production process. Inflammable and explosive problems, coupled with defects such as difficult regeneration of column chromatography materials, lead to the above patents only suitable for small batches and laboratory preparations, not suitable for industrial production.

Contents of the invention

The purpose of the present invention is to provide a method for extracting high-purity sulforaphane from broccoli seeds, so as to overcome the defect that the prior art cannot be produced industrially, so as to use the sulforaphane extracted from broccoli seeds for human prevention and treatment. Cancer lays the groundwork.

After systematic research and testing by the inventor, the method proposed comprises the following steps:

(1) Use dried broccoli seeds as raw materials, crush them and add water; hydrolyze in warm water to obtain a solid-liquid mixture;

(2) Add absolute ethanol to the hydrolyzed solid-liquid mixture, ultrasonically extract, suction filter or double-layer gauze filter;

(3) Concentrating the filtrate to evaporate ethanol under vacuum conditions;

(4) The de-alcoholized feed liquid is chromatographed on a macroporous resin column of the same polarity, and gradient elution is carried out with ethanol aqueous solution as the eluent, and the eluate containing sulforaphane is selectively collected;

(5) The obtained sulforaphane eluate is decolorized by aluminum peroxide dry column chromatography, and the sulforaphane chromatography solution is collected, then concentrated in a vacuum to an oily extract, and sulforaphane with a purity of 60% is obtained;

(6) Take the sulforaphane extract with a purity of 60% obtained in the previous step, dissolve it in water, and extract it with an organic solvent;

(7) Discard the water layer after extraction, and vacuum-concentrate the organic layer to obtain a sulforaphane extract with a purity greater than 90%.

In the above step (1), the broccoli seeds are crushed to 30-60 meshes; the mass of the added water is 1.5-2 times the mass of the seed powder; the temperature of the warm water is 25°C-35°C; the hydrolysis time For 7 ~ 9h.

In the above step (2), the mass of the added absolute ethanol is 8 to 8.5 times the mass of the solid-liquid mixture; the ultrasonic extraction time is 30 to 60 min, the ultrasonic extraction frequency is 40 kHz, and the power is 200 W.

In the above step (3), the vacuum degree during concentration under vacuum conditions is -0.06 to -0.08MPa, and the temperature is 30°C to 40°C.

In the above step (4), the models of the homopolar macroporous resins are AB-8, DM-130, D101; the mass fractions of the gradient eluted ethanol solutions are 30%, 50%, and 70%, respectively.

In the above step (5), the vacuum degree during the vacuum concentration is -0.06 to -0.08MPa, and the temperature is 30°C to 40°C.

In the above step (6), the amount dissolved in water is 10 times the amount of the extract; the organic solvent is a mixed solvent of ethyl acetate or dichloromethane and ethanol according to 1:1 composition; the extraction times are 3 times, the amount of organic solvent used in the extraction was twice the amount of the aqueous layer feed solution.

In the above-mentioned step (7), the degree of vacuum controlled during the vacuum concentration is -0.06～-0.08MPa.

The inventor pointed out that the technical steps of the present invention can be summarized as follows: Broccoli seeds are crushed to 30-60 meshes, then added 1.5-2 times of water, hydrolyzed at a temperature of 25°C-35°C for 7-9 hours, and the hydrolyzed solid-liquid mixture Add 8 to 8.5 times of absolute ethanol to the solution, then ultrasonically extract for 30 to 60 minutes, plate and frame filter (double-layer gauze), vacuum concentrate at 30°C to 40°C to remove alcohol, and use a macroporous resin column for the extract after removing alcohol Chromatography (elution gradients are 30%, 50%, and 70% ethanol aqueous solution), collect the liquid containing sulforaphane (determined by HPLC detection), and the eluent is subjected to aluminum peroxide dry column chromatography to collect the chromatographic liquid; Concentrate in vacuum at 30°C to 40°C to the extract to obtain sulforaphane with a purity of 60%. After adding an appropriate amount of water to dissolve it, extract it with an organic solvent, discard the water layer, and concentrate the organic layer in vacuum to obtain a purity greater than 90% sulforaphane extract.

The method of the invention can directly extract high-purity sulforaphane with a purity greater than 90% from broccoli seeds, and has the advantages of simple process flow, stable product quality, safe use of reagents, simple production equipment, and easy industrial production. It is suitable for enterprises that use broccoli seeds as raw materials to industrially extract sulforaphane.

Detailed ways

Example 1

Take 100g of broccoli seeds, grind them to 30-60 mesh, add 200ml of water; hydrolyze at 35°C for 8h, add 800ml of absolute ethanol, ultrasonically extract for 30min, the frequency of ultrasonic extraction is 40kHz, and the power is 200W; gauze filter;

Concentrate the filtrate in vacuum at 40°C to evaporate ethanol; use suction filtration to filter until clear, and use AB-8 macroporous resin column chromatography to remove impurities with 400mL of ethanol with a mass fraction of 30%. , and discard the eluent, then use 600mL of ethanol with a mass fraction of 50% to elute the sulforaphane on the macroporous resin chromatography column, collect the eluate, and then use alumina column chromatography to collect the sulforaphane The eluate was then vacuum-concentrated at 40°C to an oily extract, 5 mg of the concentrated oil was weighed and dissolved in 10 ml of methanol, and then detected by HPLC. The purity of sulforaphane in the product was 60%.

Dissolve the sulforaphane extract obtained in the previous step with a purity of 60% in an appropriate amount of water, extract it three times with ethyl acetate, discard the water layer, collect the organic layer, and concentrate at 40°C under vacuum -0.08MPa to an oily extract; weigh 5 mg of the concentrated oil, dissolve it in 10 mL of methanol, and then detect it by HPLC to obtain a sulforaphane extract with a purity greater than 90%.

Example 2

Take 100g of broccoli seeds, grind them to 30-60 mesh, add 200mL of water; hydrolyze at 35℃ for 10h, add 800mL of absolute ethanol, ultrasonically extract for 30min, ultrasonic extraction frequency 40kHz, power 200W; gauze filter;

The filtrate was vacuum-concentrated and evaporated ethanol at 40°C; it was filtered until clear by suction filtration, and 150 mL of the clear feed liquid was chromatographed on a DM130 macroporous resin column, and impurities were removed with 400 mL of ethanol with a mass fraction of 30%, and Discard the eluate, and then use 600mL of ethanol with a mass fraction of 50% to elute the sulforaphane on the macroporous resin chromatography column, collect the eluate, and then use alumina column chromatography, and collect the sulforaphane eluted liquid, and then vacuum-concentrated at 40°C to an oily extract, weighed 5mg of the concentrated oily substance and dissolved it in 10mL of methanol, and then detected by HPLC, the purity of sulforaphane in the product was 60%.

Dissolve the 60% pure sulforaphane extract obtained in the previous step in an appropriate amount of water, extract three times with a 1:1 mixed solvent of dichloromethane and ethanol, collect the organic layer, and concentrate it at 40°C under vacuum -0.06MPa to For the oily extract, 5 mg of the concentrated oil was weighed and dissolved in 10 mL of methanol, and then detected by HPLC to obtain a sulforaphane extract with a purity greater than 90%.

Claims (8)

1. method of from broccoli seed, extracting high-purity sulforaphen, its feature may further comprise the steps:

⑴ be raw material with dry broccoli seed, adds water after the pulverizing; In warm water, be hydrolyzed, obtain solidliquid mixture;

⑵ add dehydrated alcohol in the solidliquid mixture after the hydrolysis, ultrasonic lixiviate, suction filtration or double gauze filter;

⑶ with filtrate concentration and evaporation ethanol under vacuum condition;

⑷ will remove the rear feed liquid homopolarity macroporous resin column chromatography of alcohol, be that eluent carries out gradient elution with aqueous ethanolic solution, and selective collection contains the sulforaphen elutriant;

⑸ will obtain the sulforaphen elutriant, and the sulforaphen chromatographic solution is collected in peroxidation aluminium dry chromatography decolouring, again through vacuum concentration to oily medicinal extract, namely obtain purity and be 60% sulforaphen;

(6) being obtained purity the upper step is 60% sulforaphen medicinal extract, after being dissolved in water, adopts organic solvent to extract;

(7) discard water layer after the extraction, organic layer carries out vacuum concentration, namely gets purity greater than 90% sulforaphen extract.

2. in accordance with the method for claim 1, it is characterized in that described broccoli seed is crushed to 30～60 orders in the step (1); The described quality that adds water is 1.5～2 times of seed meal quality; The temperature of described warm water is 25 ℃～35 ℃; Described hydrolysis time is 7～9h.

3. in accordance with the method for claim 1, it is characterized in that in the step (2) that the quality of described adding dehydrated alcohol is 8～8.5 times of solidliquid mixture quality; Described ultrasonic extraction time is 30～60 minutes, ultrasonic lixiviate frequency 40kHz, power 200W.

4. in accordance with the method for claim 1, it is characterized in that in the step (3), the vacuum tightness when concentrated under the described vacuum condition be-0.06 MPa extremely-0.08MPa, temperature is 30 ℃～40 ℃.

5. in accordance with the method for claim 1, it is characterized in that in the step (4) that described homopolarity macroporous resin is a kind of among AB-8, DM-130, the D101; The ethanolic soln concentration of described gradient elution is respectively 30%, 50%, 70%.

6. in accordance with the method for claim 1, it is characterized in that in the step (5) that the vacuum tightness during described vacuum concentration is-0.06 MPa to-0.08MPa, temperature is 30 ℃～40 ℃.

7. in accordance with the method for claim 1, it is characterized in that in the step (6) that the described amount that is dissolved in water is 10 times of amounts of medicinal extract amount; Described organic solvent is ethyl acetate or methylene dichloride and ethanol according to the mixed solvent that forms at 1: 1; Described extraction times is 3 times, and the amount that extracts used organic solvent is the doubling dose of water layer feed liquid.

8. in accordance with the method for claim 1, it is characterized in that in the step (7), the vacuum tightness of controlling during described vacuum concentration is-0.06～-0.08MPa.