CN114807258A - Method for extracting ceramide from pith of Malus micromalus Linn - Google Patents
Method for extracting ceramide from pith of Malus micromalus Linn Download PDFInfo
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- CN114807258A CN114807258A CN202210385862.5A CN202210385862A CN114807258A CN 114807258 A CN114807258 A CN 114807258A CN 202210385862 A CN202210385862 A CN 202210385862A CN 114807258 A CN114807258 A CN 114807258A
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- 229940106189 ceramide Drugs 0.000 title claims abstract description 86
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 title claims abstract description 85
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 title claims abstract description 85
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 title claims abstract description 85
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 46
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- IINNWAYUJNWZRM-UHFFFAOYSA-L erythrosin B Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 IINNWAYUJNWZRM-UHFFFAOYSA-L 0.000 description 2
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- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
- C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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Abstract
The invention discloses a method for extracting ceramide from seal rice bran, belonging to the technical field of separation and purification. The method comprises the following steps: performing double-enzyme enzymolysis on rice bran by sphingomyelinase and cellulase, filtering, soaking in ethanol solution, performing ultrasonic heat extraction, extracting with petroleum ether to obtain ceramide extract, and performing silica gel column chromatography and SPE small column chromatography on the ceramide extract to obtain ceramide. The invention realizes the high-efficiency extraction and purification of ceramide in the cervus chinensis rice bran by a double-enzyme composite enzymolysis method and combining ultrasonic heat extraction and SPE column chromatography technology.
Description
Technical Field
The invention belongs to the technical field of enzymolysis, relates to separation and purification of functional components, and particularly relates to a method for extracting ceramide from seal rice bran.
Background
In China, rice is the main grain producing country, the rice yield per year can reach more than 1.8 hundred million tons, and rice bran accounts for 5-8% of the rice, so that the rice bran is a high-quality renewable resource, but at present, the deep development and utilization level of the rice bran is low, and most of the rice bran is treated as feed. The sea rice is a new variety of saline-alkali resistant rice growing on the seaside tidal flat, and the rice husk of the sea rice is purplish red, so the sea rice is also called as sea red rice and is plain with the reputation of medicinal rice. The byproduct of the processing of the sea red rice, the sea red rice bran is rich in nutrient components, contains functional components such as dietary fiber, protein, vitamin, polysaccharide, fatty acid, phytic acid, mineral substances and the like, and the content of various nutrient substances is far higher than that of the rice bran of the white rice; in addition, it has been found that the red rice bran is more abundant in ceramide compounds than the white rice bran.
Ceramides are formed by covalently bonding long-chain sphingosine to fatty acids via amide bonds, where the carbon chain length, saturation and number of hydroxyl groups of sphingosine and fatty acids can vary. In recent years, with research and development on the biological activity of ceramide, ceramide becomes an important active ingredient for functional medical health products, foods, cosmetics and the like, and has a very great application value. However, due to the diversity of the molecular structure of ceramide and the very low content of ceramide in biological samples, extraction of natural ceramide is difficult. Therefore, in recent years, various researchers and researchers have been studying to separate and extract such compounds from various animals and plants, and among them, extraction of ceramide from natural plants has a wide prospect due to high safety and low cost.
CN106008253A discloses a method for extracting high-purity ceramide from pith of marine red rice, which comprises (1) pretreatment: removing impurities from the raw material of the cervus japonicus, crushing, sieving, performing enzymolysis, and filtering to obtain enzymolysis rice bran; (2) microwave countercurrent extraction: adding an organic solvent into the enzymatic rice bran, performing microwave countercurrent extraction, and performing heat preservation and filtration to obtain a rice bran extract; (3) concentration: concentrating the rice bran extract and recovering the organic solvent to obtain a rice bran concentrate; (4) and (3) organic solvent extraction and separation: stirring and extracting the rice bran concentrated solution with an organic solvent, and concentrating under reduced pressure to obtain a tar-like lipid mixture; (5) carrying out silica gel chromatography adsorption separation, eluting with an organic solvent, and collecting ceramide target fraction; (6) concentrating and drying to obtain the ceramide product. However, the patent adopts a single enzymolysis method, has low extraction efficiency of ceramide, is not thorough in extraction, is greatly influenced by environmental factors, and has an unsatisfactory actual application effect on the rice bran with high ceramide content, such as the red blood rice bran.
The extraction of ceramide from the marine red rice bran is a very effective way to utilize the marine red rice bran resource. The existing extraction method mainly comprises 95% ethanol reflux and water reflux extraction, wherein the 95% ethanol extraction method has high production cost and low operability; the water reflux extraction of the crude product has too many impurities, which greatly affects the subsequent purification process and causes difficult separation and purification. On the other hand, the existing method is not suitable for extracting the rice bran with higher ceramide content, such as the red blood rice bran. Therefore, the method for efficiently extracting the ceramide from the sea red rice bran is provided, and the sea red rice bran resource is expected to be effectively utilized.
Disclosure of Invention
The invention aims to solve the problems of insufficient extraction of ceramide from the erythrosine bran, low yield and the like in the existing extraction method, and provides a reference for effectively utilizing erythrosine bran resources and similar plants to extract ceramide.
In view of the above, the present invention addresses this need in the art by providing a process for extracting ceramide from the rice bran of Haihong.
In one aspect, the invention relates to a method for extracting ceramide from marine red rice bran, which comprises the following steps: performing double-enzyme enzymolysis on rice bran by sphingomyelinase and cellulase, filtering, soaking in ethanol solution, performing ultrasonic heat extraction, extracting with petroleum ether to obtain ceramide extract, and separating and purifying the ceramide extract by silica gel column chromatography and SPE small column to obtain ceramide.
Further, in the method for extracting ceramide from the marine red rice bran provided by the invention, the enzymatic hydrolysis comprises: preparing the sphingomyelinase and the cellulase into an enzymolysis solution with the concentration of 0.2-0.5 w/v%; the ratio of the rice bran to the enzymolysis liquid is 1: 3-5 in g: mL; the ratio of the sphingomyelinase to the cellulase is 4:6 in terms of enzyme activity unit.
Further, in the method for extracting ceramide from the marine red rice bran, the concentration of the enzymolysis liquid is 0.4 w/v%.
Further, in the method for extracting ceramide from the marine red rice bran, the enzymolysis temperature is 35-45 ℃, the time is 60-120 min, and the pH value is 5-7.
Further, in the method for extracting ceramide from the marine red rice bran, the concentration of the ethanol solution is 75-95%, and the soaking time is 1 h; the ratio of the product obtained by filtering after the double-enzyme enzymatic hydrolysis to the ethanol solution is 1:5 in g: mL; the power of ultrasonic heat extraction is 360w, the temperature is 45 ℃, and the extraction time is 1 h.
Further, in the method for extracting ceramide from the marine red rice bran provided by the invention, before petroleum ether extraction, a product obtained by ultrasonic heat extraction needs to be filtered, evaporated and concentrated to a concentrated solution with the ethanol concentration of 10%; the petroleum ether extraction comprises: adding 2 times of petroleum ether into the concentrated solution, stirring for 0.5h, standing for 0.5h, collecting the upper layer of ether phase, extracting for 2 times, combining the ether phases, and recovering the petroleum ether to obtain the ceramide extract.
Further, in the method for extracting ceramide from the marine red rice bran provided by the invention, the silica gel column chromatography comprises the following steps: performing wet column packing by using 300-400-mesh silica gel, wherein the ratio of the sample loading amount of the ceramide extract to the silica gel is 1-5: 100 in terms of mass ratio; performing gradient elution by using petroleum ether and ethyl acetate according to the proportion of 95:5, 85:15, 60:40 and 40:60 in a volume ratio, wherein the use amount of the proportion of 95:5 and the proportion of 85:15 are respectively 1BV, the use amount of the proportion of 60:40 and the proportion of 40:60 are respectively 2BV, the elution speed is 2-4 BV/h, collecting effluent liquid step by step, detecting the effluent liquid by TLC, combining the effluent liquid with the same components, recovering the solvent and drying.
Further, in the method for extracting ceramide from the marine red rice bran provided by the invention, the SPE cartridge comprises: eluting a product obtained by the silica gel column chromatography with acetone, wherein the ratio of the product obtained by the silica gel column chromatography to the acetone is 1:2 in terms of g: mL, and then dissolving the product in a wet method to load the product with the sample loading amount of 0.1-0.5 g; according to the volume ratio, ethyl acetate and acetone are used for gradient elution according to the proportion of 6:4 and 1:1, the using amount of the ethyl acetate and the acetone is 2-2.5 BV, the elution speed is 1-1.5 BV/h, the working pressure is 5-20 bar, effluent liquid is collected step by step, TLC is used for detecting the effluent liquid, the effluent liquid with the same components is combined, the solvent is recovered, and the ceramide is obtained through drying.
The invention adopts sphingomyelinase and cellulase to carry out double-enzyme enzymolysis, combines ethanol and ultrasonic heat extraction, then carries out petroleum ether extraction, and then carries out silica gel column chromatography and SPE small column purification, thus realizing the high-efficiency extraction of ceramide from rice bran. Thus, the invention further claims the use of the above process for extracting ceramide, said rice bran comprising red rice bran; and ceramide prepared by the method.
Compared with the prior art, the invention has the following beneficial effects or advantages:
the method provided by the invention is based on the sphingomyelinase and cellulase to carry out double-enzyme enzymolysis, and combines the ultrasonic heat extraction technology, so that the full extraction of the ceramide in the marine red rice bran is realized, and the purity of the obtained rice bran extract ceramide product is more than or equal to 95 percent and is obviously higher than that and the extraction rate of the rice bran extract ceramide obtained in the prior art. In addition, the invention adopts silica gel column chromatography and SPE column chromatography to prepare high-purity ceramide, and has the characteristics of convenient extraction, short period and high extraction rate and purity of the purified ceramide.
Drawings
FIG. 1 is a process flow diagram of the present invention for preparing purified ceramide from the marine red rice bran.
Detailed Description
The following examples are given to illustrate the technical aspects of the present invention, but the present invention is not limited to the following examples.
The red rice bran raw material used in the embodiment of the invention is produced in Zhanjiang Guangdong province; the cellulase (CAS No: 9012-54-8) and sphingomyelinase (CAS No: 9031-54-3) used were purchased from Kyoho technology Co., Ltd; the specification of the used chromatographic column is phi 40 multiplied by 300mm, and the material of the chromatographic column is stainless steel; the silica gel adsorbent used is purchased from Shanghai Yao trade Co., Ltd; SPE cartridges were purchased from Waters; other chemicals and equipment used, unless otherwise specified, were obtained from conventional commercial sources.
Example 1
This example provides an application test of extracting ceramide from the marine red rice bran by using a two-enzyme enzymolysis combined with an ultrasonic heat extraction technique.
Removing impurities from dried rice bran raw materials of the red sea rice, crushing, sieving to obtain rice bran raw material powder of 20-40 meshes, taking 100g of the red sea rice bran, adding 300mL of enzymolysis liquid (the concentration is 0.2 w/v%) of a preparation (the ratio of sphingomyelinase to cellulase is 4:6 in terms of enzyme activity unit), adjusting the pH value to 5 at 35 ℃, performing enzymolysis for 60min, and filtering to obtain enzymolysis rice bran (containing water). Taking 1g of enzymolysis rice bran (containing water), adding 500mL of 75% ethanol solution, stirring for 20 minutes, standing, soaking for 1h, sealing, putting into an ultrasonic cleaning machine, starting the program, selecting the ultrasonic power to be 360w, heating at 45 ℃, and extracting for 1 h. And after extraction, filtering, transferring the ethanol extract of the ceramide to a rotary evaporator for concentration until the ethanol concentration is 10%, transferring the concentrated solution to a clean three-neck flat-bottom flask, adding 2 times of petroleum ether according to the volume of the concentrated solution, stirring for 0.5h, transferring to a separating funnel for standing for 0.5h, and collecting the ether phase of the upper layer for extraction for 2 times after layering. And combining ether phases, recovering petroleum ether and concentrating to obtain an extract to obtain a brown ceramide sample I with the weight of 16.4 g.
And (2) matching 1g of crude product with 100g of silica gel according to the sample amount, weighing 100g of 300-mesh silica gel, activating for 1h at 105-110 ℃, then putting the activated silica gel into a container, adding a proper amount of petroleum ether, stirring along a fixed direction, and putting the silica gel into a silica gel chromatographic column by a wet method when no bubbles exist in the silica gel, wherein the silica gel column has the diameter of 40 x 190mm, and the column volume is 250 mL. The column was then equilibrated with an initial eluent of 500mL at a flow rate of 4mL/min for 30 min.
Dissolving the crude ceramide I by using an initial mobile phase, and then carrying out wet loading on the samples, wherein the weight ratio of petroleum ether/ethyl acetate (v/v) 95: 5. 85: 15. 60: 40. 40: gradient elution with eluent 60, 95:5 and 85:15 the using amount of each of the two eluents is 1BV, the using amount of each of the two eluents is 2BV, effluent liquid is collected step by step with the eluting speed of 2BV/h, TLC is used for detecting the effluent liquid, the effluent liquid with the same components is combined, the solvent is recovered, and the ceramide sample II with the weight of 0.45g is obtained after drying.
The ceramide sample II, which was 0.1g in the amount as above, was filtered with the initial mobile phase, and wet-packed into a medium-pressure chromatography column when no air bubbles were present in the column. The column was then compressed with a compression device to a working pressure of 10bar at normal flow rate and equilibrated with the initial eluent.
According to the volume ratio, ethyl acetate and acetone are used for gradient elution according to the mixture ratio of 6:4 and 1:1, the using amount of the ethyl acetate and the acetone is 2BV, the elution speed is 1BV/h, effluent liquid is collected step by step, TLC is used for detecting the effluent liquid, the effluent liquid with the same components is combined, the solvent is recovered, and the ceramide is obtained by drying.
Example 2
This example provides an application test of extracting ceramide from the marine red rice bran by using a two-enzyme enzymolysis combined with an ultrasonic heat extraction technique.
The extraction process of this example is the same as that of example 1, except that: the concentration of the enzymolysis liquid is 0.4w/v%, the enzymolysis temperature is 40 ℃, the enzymolysis pH is 6, and the enzymolysis time is 90 min; the column chromatography of silica gel adopts 400-mesh silica gel, the ratio of the sample loading amount of the ceramide extract to the silica gel is 3:100, and the elution speed of petroleum ether and ethyl acetate is 3 BV/h; in the SPE small column, the working pressure is 5 bar.
Example 3
This example provides an application test of extracting ceramide from the marine red rice bran by using a two-enzyme enzymolysis combined with an ultrasonic heat extraction technique.
The extraction process of this example is the same as that of example 1, except that the concentration of the enzymolysis solution is 0.5w/v%, the enzymolysis temperature is 45 ℃, the enzymolysis pH is 7, the enzymolysis time is 120min, and the concentration of the ethanol solution is 95%; the ratio of the sample loading amount of the ceramide extract to the silica gel is 5:100, and the elution speed of the petroleum ether and the ethyl acetate is 4 BV/h; the dosage of the ethyl acetate and the acetone is 2.5BV, the elution speed is 1.5BV/h, and the working pressure is 20 bar.
Comparative example 1
This example provides an application test for extracting ceramide from the marine red rice bran using a single enzymatic hydrolysis process.
The extraction process of this example is the same as that of example 1, except that the enzyme used for enzymatic hydrolysis is sphingomyelinase.
Comparative example 2
This example provides an application test for extracting ceramide from the marine red rice bran using a single enzymatic hydrolysis process.
The extraction process of this example is the same as that of example 1, except that the enzyme used for enzymolysis is cellulase.
Comparative example 3
This example provides an application test for extracting ceramide from the marine red rice bran using conventional organic solvent extraction.
The extraction procedure of this example is the same as that of example 1 except that a conventional organic solvent extraction is used before silica gel column chromatography. The traditional organic solvent extraction process is as follows:
adding 100g of the sea red rice bran into 500mL of 95% ethanol solution, stirring for 30min, standing for 1h, and then performing extraction operation, wherein the condensing reflux time is 1 h. And after extraction, filtering, transferring the ethanol extract of the ceramide to a rotary evaporator for concentration until the ethanol concentration is 10%, transferring the concentrated solution to a clean three-neck flat-bottom flask, adding 2 times of petroleum ether according to the volume of the concentrated solution, stirring for 0.5h, transferring to a separating funnel for standing for 0.5h, and collecting the ether phase of the upper layer for extraction for 2 times after layering. And combining ether phases, recovering petroleum ether and concentrating to obtain an extract to obtain a brown ceramide sample I with the weight of 8.36 g.
Comparative example 4
This example provides an application test for extracting ceramide from the marine red rice bran by supercritical fluid extraction.
The extraction process of this example is the same as that of example 1, except that supercritical fluid extraction is used before silica gel column chromatography. The supercritical fluid extraction process is as follows:
adding 100g of sea red rice bran into 300mL of 95% ethanol solution, stirring and standing for 1h, then opening a supercritical fluid extraction machine, and adding CO 2 The machine is brought to a supercritical state and then an extraction operation is carried out. And after extraction, filtering, transferring the ethanol extract of the ceramide to a rotary evaporator for concentration until the ethanol concentration is 10%, transferring the concentrated solution to a clean three-neck flat-bottom flask, adding 2 times of petroleum ether according to the volume of the concentrated solution, stirring for 0.5h, transferring to a separating funnel for standing for 0.5h, and collecting the ether phase of the upper layer for extraction for 2 times after layering. The ether phases are combined, and the petroleum ether is recovered and concentrated to an extract, thus obtaining a brown ceramide sample I with the weight of 12.49 g.
Example 4
This example provides a comparative experiment of ceramide extraction using the above method.
The extraction processes of examples 1 to 3 and comparative examples 1 to 4 were compared, and the comparison results are shown in Table 1.
TABLE 1 comparison of extraction yields for different extraction modes
As can be seen from Table 1, the crude extraction rate of ceramide in the product obtained in the embodiment of the invention has obvious advantages compared with other extraction modes, the crude extraction rate of the obtained ceramide reaches 16.48 +/-0.09% at most, the quality of the obtained product is 16.4g at most, and the purity of the obtained ceramide is 96.13%.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (10)
1. A method for extracting ceramide from seal rice bran, which is characterized by comprising the following steps: performing double-enzyme enzymolysis on rice bran by sphingomyelinase and cellulase, filtering, soaking in ethanol solution, performing ultrasonic heat extraction, extracting with petroleum ether to obtain ceramide extract, and separating and purifying the ceramide extract by silica gel column chromatography and SPE small column to obtain ceramide.
2. The method of claim 1, wherein the enzymatic hydrolysis comprises: preparing the sphingomyelinase and the cellulase into an enzymolysis solution with the concentration of 0.2-0.5 w/v%;
the ratio of the rice bran to the enzymolysis liquid is 1: 3-5 in g: mL;
the ratio of the sphingomyelinase to the cellulase is 4:6 in terms of enzyme activity unit.
3. The method for extracting ceramide from marine red rice bran according to claim 2, wherein the concentration of the enzymatic hydrolysate is 0.4 w/v%.
4. The method for extracting ceramide from marine red rice bran according to claim 2, wherein the enzymolysis temperature is 35-45 ℃, the time is 60-120 min, and the pH is 5-7.
5. The method for extracting ceramide from marine red rice bran according to claim 1, wherein the concentration of the ethanol solution is 75% -95%, and the soaking time is 1 h; the ratio of the product obtained by filtering after the double-enzyme enzymatic hydrolysis to the ethanol solution is 1:5 in g: mL;
the power of ultrasonic heat extraction is 360w, the temperature is 45 ℃, and the extraction time is 1 h.
6. The method for extracting ceramide from marine red rice bran according to claim 1, wherein the product obtained by the ultrasonic heat extraction is filtered, evaporated and concentrated to a concentrated solution with 10% ethanol concentration before petroleum ether extraction;
the petroleum ether extraction comprises: adding 2 times of petroleum ether into the concentrated solution, stirring for 0.5h, standing for 0.5h, collecting the upper layer of ether phase, extracting for 2 times, combining the ether phases, and recovering the petroleum ether to obtain the ceramide extract.
7. The method of claim 1, wherein the silica gel column chromatography comprises: performing wet column packing by using 300-400-mesh silica gel, wherein the ratio of the sample loading amount of the ceramide extract to the silica gel is 1-5: 100 in terms of mass ratio;
performing gradient elution by using petroleum ether and ethyl acetate according to the proportion of 95:5, 85:15, 60:40 and 40:60 in a volume ratio, wherein the use amount of the proportion of 95:5 and the proportion of 85:15 are respectively 1BV, the use amount of the proportion of 60:40 and the proportion of 40:60 are respectively 2BV, the elution speed is 2-4 BV/h, collecting effluent liquid step by step, detecting the effluent liquid by TLC, combining the effluent liquid with the same components, recovering the solvent and drying.
8. The method of claim 1, wherein said SPE cartridge comprises: eluting a product obtained by the silica gel column chromatography with acetone, wherein the ratio of the product obtained by the silica gel column chromatography to the acetone is 1:2 in terms of g: mL, and then dissolving the product in a wet method to load the product with the sample loading amount of 0.1-0.5 g; according to the volume ratio, ethyl acetate and acetone are used for gradient elution according to the proportion of 6:4 and 1:1, the using amount of the ethyl acetate and the acetone is 2-2.5 BV, the elution speed is 1-1.5 BV/h, the working pressure is 5-20 bar, effluent liquid is collected step by step, TLC is used for detecting the effluent liquid, the effluent liquid with the same components is combined, the solvent is recovered, and the ceramide is obtained through drying.
9. Use of the method of any one of claims 1 to 8 for extracting ceramide from a marine red rice bran, including marine red rice bran.
10. A ceramide prepared by the method for extracting ceramide from the marine red rice bran according to any one of claims 1 to 8.
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