CN108440356B - Method for extracting sulforaphene from radish seeds - Google Patents
Method for extracting sulforaphene from radish seeds Download PDFInfo
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- CN108440356B CN108440356B CN201810642564.3A CN201810642564A CN108440356B CN 108440356 B CN108440356 B CN 108440356B CN 201810642564 A CN201810642564 A CN 201810642564A CN 108440356 B CN108440356 B CN 108440356B
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- enzymolysis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
- C07C331/16—Isothiocyanates
- C07C331/18—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms
- C07C331/22—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
Abstract
The invention discloses a method for extracting sulforaphene from radish seeds, which comprises the following steps: adding water into radish seeds, and then carrying out heat preservation enzymolysis pretreatment to obtain an enzymolysis product; and (3) extracting the enzymolysis product by using an organic solvent, and then removing the solvent to obtain the sulforaphene extract. According to the technical scheme provided by the invention, radish seeds do not need to be subjected to degreasing treatment, the use and recovery of a solvent are greatly reduced, the water consumption is low, meanwhile, the radish seeds are subjected to heat preservation enzymolysis pretreatment and then are extracted by a solvent extraction method, so that the radish can be effectively extracted, and the method has the advantages of low water consumption, short enzymolysis time and short extraction time in the pretreatment process, and is simpler and more convenient in extraction process.
Description
Technical Field
The invention relates to the technical field of extraction and purification, and particularly relates to a method for extracting sulforaphene from radish seeds.
Background
Radish seeds, also known as radish seeds, are the mature seeds of radish of the crucifer family. Cruciferous plants and their related plant families are rich in secondary plant metabolites, including over 120 glucosinolates and various isothiocyanates, which are widely used by humans for cancer prevention, oxidation resistance, and antibacterial purposes. Sulforaphane (SFE) and Sulforaphane (SFA) are the isothiocyanates that have been found to date to have the best anticancer activity. SFA is a cancer preventative agent found in broccoli. SFE has one more double bond than SFA in structure, and related research shows that under a certain dosage concentration of sulforaphane, the compound can reduce the proliferation of cancer cells and induce apoptosis, such as colon cancer cell lines, human and murine leukemia cells, human T lymphocytes and human cervical carcinoma cells.
The sulforaphane is the main sulfur-containing compound in radish seeds, and the broccoli and the purple cabbage also contain a small amount of sulforaphane. Relevant research shows that the sulforaphene can induce apoptosis of human lung adenocarcinoma cells A549 by inhibiting tubulin polymerization. Related reports suggest that, within a certain dosage range, sulforaphane inhibits cell proliferation of K562, HT-29 and colon cancer cells. Reducing proliferation of human and murine erythroleukemia cell lines, human T lymphocytes, human cervical cancer cells, and the H3-T1-1 cell line. In addition, for in vitro resistance to mutagenicity, the effect of sulforaphane on A1254 cells in mouse liver is higher than that of SFA with excellent anticancer activity at present.
At present, radish seeds are mostly added into a large amount of phosphate buffer solution or water for enzymolysis, then the pH value is adjusted to be lower, most of protein is denatured, enzymolysis liquid containing the radish is obtained by filtration, and then a crude extract of the radish is obtained by liquid-liquid extraction and concentration methods. However, the whole process generates a large amount of waste water, which increases the production cost and the use of equipment; meanwhile, radish seed resources are greatly wasted, and comprehensive utilization of radish seeds is not facilitated.
Disclosure of Invention
The invention mainly aims to provide a method for extracting sulforaphene from radish seeds, aiming at reducing the amount of waste water generated in the technical process of extracting the sulforaphene from the radish seeds.
In order to achieve the purpose, the invention provides a method for extracting sulforaphene from radish seeds, which comprises the following steps:
adding water into radish seeds, and then carrying out heat preservation enzymolysis pretreatment to obtain an enzymolysis product;
and (3) extracting the enzymolysis product by using an organic solvent, and then removing the solvent to obtain the sulforaphene extract.
Preferably, the method comprises the following steps of adding water into radish seeds, and then carrying out heat preservation enzymolysis pretreatment to obtain an enzymolysis product: the temperature of the heat preservation enzymolysis pretreatment is 4-40 ℃, and the time is 5-40 min.
Preferably, the method comprises the following steps of adding water into radish seeds, and then carrying out heat preservation enzymolysis pretreatment to obtain an enzymolysis product: the temperature of the heat preservation enzymolysis pretreatment is 15-40 ℃, and the time is 10-30 min.
Preferably, the method comprises the following steps of adding water into radish seeds, and then carrying out heat preservation enzymolysis pretreatment to obtain an enzymolysis product: the addition amount of the water is 0.1-4.8 times of the mass of the radish seeds.
Preferably, the step of extracting the enzymolysis product with an organic solvent and then removing the solvent to obtain the sulforaphene extract comprises the following steps: the organic solvent is any one of dichloromethane, acetonitrile, chloroform, diethyl ether and ethyl acetate.
Preferably, the step of extracting the enzymolysis product with an organic solvent and then removing the solvent to obtain the sulforaphene extract comprises the following steps: the solvent removal method is rotary evaporation, reduced pressure distillation or vacuum desolventization.
According to the technical scheme provided by the invention, the whole radish seeds are directly used as the extraction raw material, degreasing treatment is not needed, the use and recovery of a solvent are greatly reduced, the water consumption is low, meanwhile, heat-preservation enzymolysis pretreatment is firstly carried out on the radish seeds, and then the radish element in the radish seeds is extracted by a solvent extraction method, so that the radish element can be effectively extracted, and the method has the advantages of low water consumption, short enzymolysis time and short extraction time in the pretreatment process, and the extraction process is simpler and more convenient.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Radish seeds are rich in secondary plant metabolites including more than 120 glucosinolates and various isothiocyanates, which can be hydrolyzed by myrosinase, an endogenous enzyme released from plant cells, to mainly produce isothiocyanates including sulforaphene (sulforaphene) which is widely used by humans in anticancer and cancer prevention, anti-oxidation and antibacterial aspects, and some nitrogen and sulfur-containing compounds. Most of the current natural sulforaphane is prepared by adding radish seeds into a large amount of phosphate buffer solution or water for enzymolysis, for example, the radish seeds are crushed and degreased, the degreased residue is added with water for enzymolysis for 1h, the enzymolysis temperature is 25 ℃, the enzymolysis environment pH is 7.0, the liquid-solid ratio is 20:1, and then the radish seeds are extracted by an organic solvent for 5min, the extraction temperature is 25 ℃, so that a sulforaphane extract is obtained; for another example, the radish seed or radish seed sprout is pulverized or homogenized, added with 5-20 times volume of water or buffer solution with pH value of 5.0-8.0, stirred and hydrolyzed at 5-50 ℃ for 10-300 min, added with hydrochloric acid to adjust the pH value of the hydrolysate to 1.0-3.0, kept standing overnight, subjected to reduced pressure filtration or centrifugation to obtain a radish seed hydrolysate, and subjected to organic solvent extraction and solvent recovery to obtain a dried radish seed extract. The two methods generate a large amount of waste water in the whole process, and increase the production cost and the use of equipment.
In order to solve the problem of large amount of waste water generated by the existing technological method for extracting the sulforaphene, the invention provides a method for extracting the sulforaphene from radish seeds, which comprises the following steps:
step S10, adding water into the radish seeds, and then carrying out heat preservation enzymolysis pretreatment to obtain an enzymolysis product;
in the embodiment of the invention, the adopted radish seeds are the whole radish seeds, and the whole radish seeds are directly taken for heat preservation enzymolysis pretreatment without carrying out degreasing treatment on the radish seeds, so that the use and recovery of a solvent generated by a radish seed degreasing treatment process are avoided, and the water consumption is reduced.
The specific implementation method comprises the following steps: adding water into the radish seeds, and then carrying out heat preservation and enzymolysis for 5-40 min at the temperature of 4-40 ℃ to obtain an enzymolysis product. In the heat preservation enzymolysis process, glucosinolate in the radish seeds is hydrolyzed by myrosinase released by the radish seed cells to generate sulforaphene in an environment in contact with water, so that the content of the sulforaphene is further improved, the heat preservation is carried out for 5-40 min, the enzymolysis can be complete, and compared with the prior art that the pH value needs to be adjusted and the enzymolysis solution is placed overnight during enzymolysis, the method has the advantages of short enzymolysis time, no need of adjusting the pH value of the enzymolysis solution, and simpler and more convenient process.
The heat preservation enzymolysis process can be carried out in an ice water bath, a constant temperature water bath or a constant temperature box according to the specific set temperature. As a preferred embodiment, the temperature of the heat preservation enzymolysis is 15-40 ℃, and the time is 10-30 min.
Meanwhile, because the sulforaphene is unstable and easy to decompose in water, excessive water addition amount often causes great decomposition of the sulforaphene, the difficulty of a subsequent separation process is increased, and a great amount of wastewater is generated.
And step S20, extracting the enzymolysis product by using an organic solvent, and removing the solvent to obtain the sulforaphene extract.
Wherein the organic solvent is any one of dichloromethane, acetonitrile, chloroform, diethyl ether and ethyl acetate. Any one of the organic solvents is used as an extraction solvent, the enzymolysis product is extracted, the generated sulforaphene is extracted, during specific operation, the extraction is carried out at room temperature, the addition amount and the extraction time of the organic solvent are not particularly limited, and can be determined according to the amount of the enzymolysis product obtained during actual operation, for example, 480g of water is added into 100g of radish seeds for carrying out heat preservation and enzymolysis on the enzymolysis product, and 200mL of the organic solvent can be added into the radish seeds for extraction for 30 min; for another example, an enzymatic hydrolysate obtained by adding 10g of water to 100g of radish seeds and performing enzymatic hydrolysis under incubation may be extracted with 100mL of an organic solvent for 25 min.
Extracting with organic solvent to obtain extractive solution, and removing organic solvent to obtain sulforaphene extract. The method for removing the organic solvent in the extract liquor can adopt rotary evaporation, reduced pressure distillation or vacuum desolventizing, the rotary evaporation and the vacuum desolventizing can be carried out by adopting a rotary evaporator and a vacuum desolventizing machine, and the reduced pressure distillation can be carried out by constructing a reduced pressure distillation device by adopting a common laboratory instrument. Preferably, the dried sulforaphene extract is obtained directly by removing the solvent using rotary evaporation.
According to the technical scheme provided by the invention, the whole radish seeds are directly used as the extraction raw material, degreasing treatment is not needed, the use and recovery of a solvent are greatly reduced, the water consumption is low, meanwhile, heat-preservation enzymolysis pretreatment is firstly carried out on the radish seeds, and then the radish element in the radish seeds is extracted by a solvent extraction method, so that the radish element can be effectively extracted, and the method has the advantages of low water consumption, short enzymolysis time and short extraction time in the pretreatment process, and the extraction process is simpler and more convenient.
The technical solutions of the present invention are further described in detail with reference to the following specific examples, which should be understood as merely illustrative and not limitative.
Example 1
(1) Taking 100g of radish seeds, adding 10g of water, and carrying out heat preservation and enzymolysis for 5min in an ice-water bath at 4 ℃ to obtain an enzymolysis product;
(2) adding 50mL of dichloromethane into the enzymolysis product, extracting at room temperature for 30min, removing solvent by rotary evaporation to obtain dried sulforaphene extract, and detecting by High Performance Liquid Chromatography (HPLC) to obtain sulforaphene extract with sulforaphene content of 0.408mg/g whole seed (calculated according to oil content of 40% of radish seed).
Example 2
(1) Taking 100g of radish seeds, adding 10g of water, and carrying out heat preservation and enzymolysis in an ice-water bath at 4 ℃ for 40min to obtain an enzymolysis product;
(2) adding 100mL of diethyl ether into the enzymolysis product, extracting at room temperature for 25min, removing solvent by vacuum distillation to obtain dried sulforaphane extract, and detecting by High Performance Liquid Chromatography (HPLC) to obtain sulforaphane extract with sulforaphane content of 0.375mg/g whole seed (calculated according to oil content of 40% of radish seed).
Example 3
(1) Taking 100g of radish seeds, adding 40g of water, and carrying out heat preservation and enzymolysis in a water bath at 25 ℃ for 10min to obtain an enzymolysis product;
(2) adding 150mL of acetonitrile into the enzymolysis product, extracting at room temperature for 35min, removing the solvent by rotary evaporation to obtain a dried sulforaphene extract, and detecting by High Performance Liquid Chromatography (HPLC) to obtain a sulforaphene extract with a sulforaphene content of 2.100mg/g of whole seeds (calculated according to an oil content of 40% of radish seeds).
Example 4
(1) Taking 100g of radish seeds, adding 480g of water, and carrying out heat preservation and enzymolysis in a water bath at 25 ℃ for 10min to obtain an enzymolysis product;
(2) adding 200mL of chloroform into the enzymolysis product, extracting at room temperature for 30min, removing the solvent by rotary evaporation to obtain a dried sulforaphene extract, and detecting by High Performance Liquid Chromatography (HPLC) to obtain the sulforaphene extract with a sulforaphene content of 4.310mg/g of whole seeds (calculated according to an oil content of 40% of the radish seeds).
Example 5
(1) Taking 100g of radish seeds, adding 400g of water, and carrying out heat preservation and enzymolysis for 20min in a thermostat at 15 ℃ to obtain an enzymolysis product;
(2) adding 50mL of ethyl acetate into the enzymolysis product, extracting at room temperature for 30min, and then performing vacuum desolventizing to obtain a dried sulforaphen extract, wherein the sulforaphen content of the sulforaphen extract is 1.832mg/g of whole seeds (calculated according to the oil content of the radish seeds of 40%) by detecting through High Performance Liquid Chromatography (HPLC).
Example 6
(1) Taking 100g of radish seeds, adding 300g of water, and carrying out heat preservation and enzymolysis for 30min in a thermostat at 40 ℃ to obtain an enzymolysis product;
(2) adding 100mL of dichloromethane into the enzymolysis product, extracting at room temperature for 30min, removing solvent by rotary evaporation to obtain dried sulforaphane extract, and detecting by High Performance Liquid Chromatography (HPLC) to obtain sulforaphane extract with sulforaphane content of 3.975mg/g whole seed (calculated according to oil content of 40% of radish seed).
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (2)
1. A method for extracting sulforaphene from radish seeds is characterized by comprising the following steps:
taking 100g of radish seeds, adding 480g of water, and carrying out heat preservation and enzymolysis in a water bath at 25 ℃ for 10min to obtain an enzymolysis product;
adding 200mL of chloroform into the enzymolysis product, extracting at room temperature for 30min, removing the solvent by rotary evaporation to obtain a dried sulforaphene extract, and detecting by high performance liquid chromatography, wherein the sulforaphene content in the sulforaphene extract is 4.310mg/g of whole seeds according to the oil content of the radish seeds of 40%.
2. A method for extracting sulforaphene from radish seeds is characterized by comprising the following steps:
taking 100g of radish seeds, adding 300g of water, and carrying out heat preservation and enzymolysis for 30min in a thermostat at 40 ℃ to obtain an enzymolysis product;
adding 100mL of dichloromethane into the enzymolysis product, extracting at room temperature for 30min, removing the solvent by rotary evaporation to obtain a dried sulforaphane extract, and detecting by high performance liquid chromatography, wherein the sulforaphane content in the sulforaphane extract is 3.975mg/g of whole seeds according to the oil content of the radish seeds of 40%.
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