CN111777691A - Extraction method of magnolia flower polysaccharide - Google Patents
Extraction method of magnolia flower polysaccharide Download PDFInfo
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Abstract
The invention relates to a method for extracting magnolia flower polysaccharide, which comprises the following steps: continuously extracting flos Magnoliae powder with water twice under subcritical condition, mixing the extractive solutions to obtain total extractive solution, concentrating the total extractive solution, precipitating with ethanol, and centrifuging to obtain crude polysaccharide; deproteinizing the crude polysaccharide by Sevage method, decolorizing, dialyzing, and lyophilizing to obtain flos Magnoliae polysaccharide. The extraction method of flos Magnoliae polysaccharide adopts subcritical extraction mode to extract flos Magnoliae polysaccharide, and has high extraction purity. The solvent used in the subcritical extraction is water, the cost is low, the method is environment-friendly, and the extraction method is simple and easy to operate.
Description
Technical Field
The invention belongs to the technical field of preparation processes of magnolia flower polysaccharides, and particularly relates to an extraction method of the magnolia flower polysaccharides.
Background
Polysaccharides are high molecular carbohydrates formed by binding more than 10 monosaccharides by glycosidic bonds, and have relative molecular weights ranging from thousands to millions. The structural units of the polysaccharide may be linked to form a linear chain or may form a branched chain. Research shows that the polysaccharide has important biological activity in the aspects of resisting tumor, inflammation and virus, reducing blood sugar, resisting aging, resisting coagulation, promoting immunity and the like. The natural plant polysaccharide has high nutrition and health care value, stable and palatable tissue structure, delicate appearance and unique taste due to the nature, health care value and unique water-soluble colloid characteristic. In addition, the natural plant polysaccharide is safe to human bodies and has high absorption and utilization rate. Glucomannan in konjak is soluble dietary fiber with highest hydration viscosity discovered by human beings so far, and has health care functions of satiety, blood fat reduction, blood sugar reduction, constipation relief and the like. Therefore, the effective development and utilization of the plant polysaccharide are beneficial to improving the life quality of human beings, improving the health level of human beings, enriching the food resources of human beings and creating more economic values for the medicine and food industries.
Flos Magnoliae is the flower bud of Magnolia biondii of Magnoliaceae, is a common Chinese medicine, and has effects of dispelling pathogenic wind and cold and relieving stuffy nose. The traditional Chinese medicine composition is clinically used for treating symptoms such as wind-cold headache, nasal obstruction, nasosinusitis, thick nasal discharge and the like. In recent years, active substance research of Chinese medicine magnolia flower mostly focuses on volatile oil, flavone and alkaloid substances, but research reports on magnolia flower polysaccharide are less. The documents report that the magnolia flower crude polysaccharide has better antioxidant capacity and bacteriostatic activity. In a certain concentration range, the magnolia flower crude polysaccharide has certain scavenging capacity on free radicals and is positively correlated with the concentration; the flos Magnoliae crude polysaccharide has certain inhibitory effect on Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Aspergillus niger and Aspergillus fumigatus.
Subcritical water is also called super heated water, high-pressure hot water or hot liquid water, and means that water is heated to a high temperature of more than 100 ℃ and below 374 ℃ critical temperature under certain pressure, and a water body is still kept in a liquid state. The hydrogen bond, the ionic hydration, the ionic association, the cluster structure and the like of the microstructure of the fluid in the subcritical state are changed, so that the physical and chemical characteristics of subcritical water are greatly different from those of water at normal temperature and normal pressure in properties. In the subcritical state, with the rise of temperature, hydrogen bonds of subcritical water are opened or weakened, so that the polarity of water can be changed in a large range by controlling the temperature and pressure of subcritical water, thereby realizing continuous extraction of effective components from water-soluble components to fat-soluble components in natural products, and realizing selective extraction. In addition, because the subcritical water extraction takes cheap and pollution-free water as an extractant, the subcritical water extraction technology is regarded as a revolutionary technology which is green, environment-friendly and wide in prospect.
The prior art discloses an extraction process of magnolia flower polysaccharide: the preparation method comprises the following steps of extracting magnolia flower dry powder with hot water, precipitating with ethanol, precipitating, redissolving with distilled water, deproteinizing, decolorizing, dialyzing with running water, collecting supernatant, concentrating, and freeze-drying to obtain magnolia flower polysaccharide [ separation and purification of magnolia flower polysaccharide, research on antibacterial and antioxidant activities, Yangxi, university of teachers and Shanxi ]. The method uses hot water for extraction, and has low extraction purity.
Disclosure of Invention
The invention aims to provide a method for extracting magnolia flower polysaccharide, and the method has the advantages of high purity of the extracted magnolia flower polysaccharide and simple operation.
In order to achieve the above purpose, the invention adopts the technical scheme that:
the extraction method of the magnolia flower polysaccharide comprises the following steps:
1) continuously extracting flos Magnoliae powder with water twice under subcritical condition, mixing the extractive solutions to obtain total extractive solution, concentrating the total extractive solution, precipitating with ethanol, and centrifuging to obtain crude polysaccharide;
2) deproteinizing the crude polysaccharide by Sevage method, decolorizing, dialyzing, and lyophilizing to obtain flos Magnoliae polysaccharide.
Further, when water is used for extraction in the step 1), the extraction temperature is 120-160 ℃, the extraction time is 3-4 hours, and the mass ratio of the magnolia flower powder to the water is 1: 15-1: 20.
Further, mixing the magnolia flower powder and water in the step 1) in a pressure reactor, heating for primary extraction to obtain primary extracting solution and primary solid residue, adding water into the primary solid residue, performing secondary extraction in the pressure reactor to obtain secondary extracting solution and secondary solid residue, and combining the primary extracting solution and the secondary extracting solution to obtain the total extracting solution.
Further, the concentration in the step 1) is to concentrate the volume of the total extracting solution to 1/4-1/5 to obtain a concentrated solution.
Further, the concentration in the step 1) is reduced pressure concentration at 45-50 ℃.
Further, the volume of the ethanol added in the step 1) is 4-5 times of the volume of the concentrated total extracting solution.
Further, the precipitation in the step 1) is carried out for 24-48 h at the temperature of 0-4 ℃.
Further, deproteinizing the crude polysaccharide in the step 2) by a Sevage method for 4-6 times.
Further, the decolorization in the step 2) is carried out by adopting D101 macroporous resin.
Further, in the step 2), 3500Da dialysis bags are adopted for dialysis for 24-48 h.
Analyzing the specific components of the flos Magnoliae refined polysaccharide by cellulose column chromatography and phenol-sulfuric acid colorimetry. The method comprises the following steps:
separating the magnolia flower polysaccharide by using cellulose DEAE-52 column chromatography, wherein the column parameters are as follows: 2.6 cm by 30 cm, eluent: 0, 0.1, 0.3, 0.5M NaCl phosphate buffer, 0.02 mol/L, pH =6.8, flow rate: 1 mL/min. Collecting the effluent in 1 tube per 10 mL, and determining polysaccharide content by phenol-sulfuric acid colorimetric method, wherein alpha-glucose is used as reference.
The average molecular weights of the three components in the magnolia flower polysaccharide are detected by Gel Permeation Chromatography (GPC). The method comprises the following steps:
the volume of 1mg of dried magnolia polysaccharide sample is determined to be 5 mL by GPC liquid phase method, the mobile phase is 0.02M sodium phosphate buffer solution, the pH of the sodium phosphate buffer solution is 6.8, the flow rate is 1 mL/min, the sample injection amount is 30 mu L, and the column temperature is 30 ℃. The GPC column was Sugar KS 805 (300X 8.0 mm, Shodex, Tokyo, Japan) and the detector was a parallax refractive detector.
The invention has the beneficial effects that:
the extraction method of the magnolia flower polysaccharide adopts a subcritical extraction mode to extract the magnolia flower polysaccharide, and the extraction purity is high. The solvent used in the subcritical extraction is water, the cost is low, the method is environment-friendly, and the extraction method is simple and easy to operate.
Drawings
FIG. 1 shows the measurement results of the components in the magnolia flower polysaccharides obtained by the extraction method of magnolia flower polysaccharides in example 1.
Detailed Description
The present invention will be further described with reference to the following examples and accompanying drawings.
Example 1
The method for extracting magnolia flower polysaccharide in the embodiment comprises the following steps:
1) pouring 10 g of flos Magnoliae powder and 200mL of water into a 350 mL pressure reactor, screwing the pressure reactor, performing primary extraction, setting the reaction temperature to rise to 150 ℃ at 5 ℃/min, keeping the temperature at 150 ℃ for 3 hours until the reaction is finished, and keeping the stirring speed of 500 rpm by a magnetic stirrer all the time during the reaction. And naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 10 min at 5000 rpm of a centrifugal machine to obtain a first-stage extracting solution and first-stage solid residues. And adding 200mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/5 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into anhydrous ethanol with 4 times of volume, and the mixture is placed into a refrigerator for precipitation for 24 hours at 4 ℃. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 5 times, decolorizing with D101 macroporous resin for 4 hr, dialyzing with 3500Da dialysis bag for 24 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Example 2
1) Pouring 20 g of flos Magnoliae powder and 400 mL of water into a 800 mL pressure reactor, screwing the pressure reactor, performing primary extraction, setting the reaction temperature to rise to 150 ℃ at 5 ℃/min, keeping the temperature at 150 ℃ for 3 hours until the reaction is finished, and keeping the stirring speed of 500 rpm by a magnetic stirrer all the time during the reaction. And naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 12 min at 5000 rpm of a centrifugal machine to obtain a first-stage extracting solution and first-stage solid residues. And adding 200mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/5 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into anhydrous ethanol with 4 times of volume, and the mixture is placed into a refrigerator for precipitation for 24 hours at 4 ℃. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 5 times, decolorizing with D101 macroporous resin for 4 hr, dialyzing with 3500Da dialysis bag for 24 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Example 3
1) Pouring 50 g of flos Magnoliae powder and 1000 mL of water into a 1500 mL pressure reactor, screwing the pressure reactor, performing primary extraction, setting the reaction temperature to rise to 150 ℃ at 5 ℃/min, keeping the temperature at 150 ℃ for 3.5 hours until the reaction is finished, and keeping the stirring speed of 500 rpm by a magnetic stirrer all the time during the reaction. Naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 15 min at 5000 rpm of a centrifuge to obtain a first-stage extracting solution and first-stage solid residues. And adding 200mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/5 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into anhydrous ethanol with 4 times of volume, and is placed into a refrigerator for precipitation at 4 ℃ for 24 hours. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 5 times, decolorizing with D101 macroporous resin for 5 hr, dialyzing with 3500Da dialysis bag for 36 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Example 4
1) Pouring 100 g flos Magnoliae powder and 2000 mL water into a 3000 mL pressure reactor, screwing the pressure reactor, performing first-stage extraction, setting reaction temperature to 150 deg.C at 5 deg.C/min, maintaining 150 deg.C for 3.5 hr until the reaction is finished, and maintaining the stirring speed of 500 rpm by magnetic stirrer during the reaction. Naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 15 min at 5000 rpm of a centrifuge to obtain a first-stage extracting solution and first-stage solid residues. And adding 200mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/5 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into anhydrous ethanol with 4 times of volume, and the mixture is placed into a refrigerator for precipitation for 36 hours at 4 ℃. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 6 times, decolorizing with D101 macroporous resin for 6 hr, dialyzing with 3500Da dialysis bag for 36 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Example 5
1) Pouring 100 g flos Magnoliae powder and 2000 mL water into a 3000 mL pressure reactor, screwing the pressure reactor, performing first-stage extraction, setting reaction temperature to 150 deg.C at 5 deg.C/min, maintaining 150 deg.C for 3.5 hr until the reaction is finished, and maintaining the stirring speed of 500 rpm by magnetic stirrer during the reaction. Naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 15 min at 5000 rpm of a centrifuge to obtain a first-stage extracting solution and first-stage solid residues. And adding 200mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/5 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into anhydrous ethanol with 4 times of volume, and the mixture is placed into a refrigerator for precipitation for 36 hours at 4 ℃. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 6 times, decolorizing with D101 macroporous resin for 6 hr, dialyzing with 3500Da dialysis bag for 36 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Example 6
1) Pouring 10 g of flos Magnoliae powder and 150mL of water into a 350 mL pressure reactor, screwing the pressure reactor, performing primary extraction, setting the reaction temperature to be increased to 120 ℃ at 5 ℃/min, keeping the temperature at 120 ℃, keeping the reaction temperature for 4 hours until the reaction is finished, and keeping the stirring speed of 500 rpm by a magnetic stirrer all the time during the reaction. And naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 10 min at 5000 rpm of a centrifugal machine to obtain a first-stage extracting solution and first-stage solid residues. And adding 150mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/4 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into anhydrous ethanol with 4 times of volume, and the mixture is placed into a refrigerator for precipitation for 24 hours at 4 ℃. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 5 times, decolorizing with D101 macroporous resin for 4 hr, dialyzing with 3500Da dialysis bag for 48 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Example 7
1) Pouring 10 g of flos Magnoliae powder and 180mL of water into a 350 mL pressure reactor, screwing the pressure reactor, performing primary extraction, setting the reaction temperature to rise to 150 ℃ at 5 ℃/min, keeping the temperature at 150 ℃ for 3 hours until the reaction is finished, and keeping the stirring speed of 500 rpm by a magnetic stirrer all the time during the reaction. And naturally cooling to room temperature after the reaction is finished, and centrifuging the reaction product for 10 min at 5000 rpm of a centrifugal machine to obtain a first-stage extracting solution and first-stage solid residues. Adding 180mL of water into the primary solid residue, and placing the mixture into a pressure reactor for secondary extraction according to the conditions set by the primary extraction to obtain a secondary extracting solution and secondary solid residue. And mixing the first-stage extract and the second-stage extract to obtain a total extract. The total extract was concentrated to 1/5 volumes under reduced pressure at 45 ℃ using a rotary evaporator to give a concentrated solution. The concentrated solution is poured into 5 times volume of absolute ethyl alcohol and is put into a refrigerator for precipitation for 24 hours at 4 ℃. The precipitate was centrifuged to obtain crude polysaccharide.
2) Deproteinizing the crude polysaccharide by Sevage method for 5 times, decolorizing with D101 macroporous resin for 4 hr, dialyzing with 3500Da dialysis bag for 48 hr, and lyophilizing to obtain refined polysaccharide of flos Magnoliae.
Test example 1
The specific components of the refined magnolia flower polysaccharide obtained in example 1 were analyzed by cellulose column chromatography and phenol-sulfuric acid colorimetry. The method comprises the following steps:
separating the magnolia flower polysaccharide by using cellulose DEAE-52 column chromatography, wherein the column parameters are as follows: 2.6 cm by 30 cm, eluent: 0, 0.1, 0.3, 0.5M NaCl phosphate buffer, 0.02 mol/L, pH =6.8, flow rate: 1 mL/min. Collecting the effluent in 1 tube per 10 mL, and determining polysaccharide content by phenol-sulfuric acid colorimetric method, wherein alpha-glucose is used as reference.
As shown in FIG. 1, 3 specific fractions of flos Magnoliae polysaccharide were detected, and the chromatographic peaks were located in 0.1 and 0.3M NaCl phosphate buffer elution fractions, respectively.
Test example 2
The average molecular weights of the three components in the magnolia flower polysaccharide obtained in example 1 were determined by Gel Permeation Chromatography (GPC). The method comprises the following steps:
the volume of 1mg of dried magnolia polysaccharide sample is determined to be 5 mL by GPC liquid phase method, the mobile phase is 0.02M sodium phosphate buffer solution, the pH of the sodium phosphate buffer solution is 6.8, the flow rate is 1 mL/min, the sample injection amount is 30 mu L, and the column temperature is 30 ℃. The GPC column was Sugar KS 805 (300X 8.0 mm, Shodex, Tokyo, Japan) and the detector was a parallax refractive detector. The average molecular weights of the three components of the magnolia flower polysaccharide are detected to be 99.20 kDa, 278.69 kDa and 215.13 kDa respectively.
Claims (10)
1. The extraction method of the magnolia flower polysaccharide is characterized by comprising the following steps:
1) continuously extracting flos Magnoliae powder with water twice under subcritical condition, mixing the extractive solutions to obtain total extractive solution, concentrating the total extractive solution, precipitating with ethanol, and centrifuging to obtain crude polysaccharide;
2) deproteinizing the crude polysaccharide by Sevage method, decolorizing, dialyzing, and lyophilizing to obtain flos Magnoliae polysaccharide.
2. The extraction method of magnolia flower polysaccharide according to claim 1, wherein when the water is used for extraction in step 1), the extraction temperature is 120-160 ℃, the extraction time is 3-4 hours, and the mass ratio of the magnolia flower powder to the water is 1: 15-1: 20.
3. The extraction method of magnolia polysaccharides according to claim 1 or 2, wherein in step 1), the magnolia powder and water are mixed in a pressure reactor, then the temperature is raised for primary extraction to obtain a primary extract and a primary solid residue, water is added into the primary solid residue, then secondary extraction is carried out in the pressure reactor to obtain a secondary extract and a secondary solid residue, and the primary extract and the secondary extract are combined to obtain the total extract.
4. The extraction method of magnolia polysaccharides according to claim 1, wherein the concentration in step 1) is to concentrate the total volume of the extract to 1/4-1/5 to obtain a concentrated solution.
5. The method for extracting magnolia polysaccharides according to claim 1 or 4, wherein the concentration in step 1) is a reduced pressure concentration at 45 to 50 ℃.
6. The method for extracting magnolia polysaccharides according to claim 1, wherein the volume of ethanol added in step 1) is 4 to 5 times of the volume of the concentrated total extract.
7. The method for extracting magnolia polysaccharides according to claim 1 or 6, wherein the precipitation in step 1) is performed at 0-4 ℃ for 24-48 h.
8. The method for extracting magnolia polysaccharides according to claim 1, wherein the crude polysaccharides in step 2) are deproteinized by the Sevage method 4-6 times.
9. The method for extracting magnolia polysaccharides according to claim 1, wherein the decolorizing in step 2) is performed with D101 macroporous resin.
10. The extraction method of magnolia polysaccharides according to claim 1, wherein in the step 2), 3500Da dialysis bags are adopted for dialysis for 24-48 h.
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Application publication date: 20201016 |