CN111154005A - Combined extraction method of fucoidin - Google Patents
Combined extraction method of fucoidin Download PDFInfo
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- CN111154005A CN111154005A CN201910603474.8A CN201910603474A CN111154005A CN 111154005 A CN111154005 A CN 111154005A CN 201910603474 A CN201910603474 A CN 201910603474A CN 111154005 A CN111154005 A CN 111154005A
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
A combined extraction method of fucoidan comprises five steps, firstly, collecting brown algae, oven drying at 40-50 deg.C, pulverizing with high-speed multifunctional pulverizer, and sieving with 60-80 mesh sieve; the second step is to add 95 percent ethanol with 10 to 15 times volume for three times of degreasing, and each time lasts for about 480 and 720 minutes; thirdly, centrifuging, adding 10-15 times of 2% CaCl2 solution, performing ultrasonic extraction at 60-70 ℃, and stirring by using a high-shear emulsifying machine; fourthly, removing brown algae starch; and fifthly, adding 3-4 times volume of 95% ethanol for precipitation, drying the precipitate at 50-60 ℃ to obtain polysaccharide, measuring the monosaccharide composition of the polysaccharide by using a PMP-pre-column derivatization high performance liquid chromatography, and measuring the sulfate group content of the polysaccharide by using a barium chloride-gelatin turbidimetry method to determine that the obtained polysaccharide is fucoidan. The method is used for extracting fucoidin.
Description
Technical Field
The invention relates to a combined extraction method of fucoidin.
Background
Fucoidan is one of fucoidan, and the fucoidan includes three types, i.e., fucoidan, and alginate starch, wherein the content of the fucoidan and the alginate is relatively high, and the content of the alginate starch is low. Fucoidan, defined as a polysaccharide containing a significant amount of fucose and sulfate groups, is a fucoidan sulfate. In recent years, people have attracted much attention because of their various excellent biological activities. Common methods for extracting fucoidan include water extraction, acid extraction, enzyme extraction and ultrasonic extraction, which have the following advantages and disadvantages: the water extraction method can avoid the degradation of the fucoidin, is a common extraction method of the fucoidin, but has low yield; the acid extraction method can improve the yield of the fucoidin, but can obviously degrade the fucoidin and influence the biological activity of the fucoidin; the enzyme method has mild conditions, has little influence on the structure of the fucoidan, can also improve the yield, but has higher cost; the ultrasonic extraction method can improve the yield of the fucoidan, but the degradation of the fucoidan can be caused by too long time. Therefore, an extraction method with simple operation, low cost, high extraction rate and no influence on the structure of fucoidan is urgently needed. The invention adopts hot CaCl2Extracting fucoidin and CaCl by combining solution-ultrasonic wave-high shear emulsifying machine2The extraction of the solution can remove the algin, and the combination of the extraction can obviously improve the yield of the algin. Meanwhile, the molecular weight difference between fucoidan and algin (the molecular weight of the algin is large and is usually 10 orders of magnitude)4-105And has a high molecular weight, the molecular weight of the brown algae starch is usually 103-104) And the fucoidin purity can be improved by removing the fucoidin through ultrafiltration.
Disclosure of Invention
The invention aims to provide a combined extraction method which can increase the yield of fucoidan, improve the purity of the fucoidan and ensure the structural integrity of the fucoidan to the greatest extent.
The above purpose is realized by the following technical scheme:
a combined extraction method of fucoidan comprises five steps, firstly, collecting brown algae, oven drying at 40-50 deg.C, pulverizing with high-speed multifunctional pulverizer, and sieving with 60-80 mesh sieve; the second step is to add 95 percent ethanol with 10 to 15 times volume for three times of degreasing, and each time lasts for about 480 and 720 minutes; thirdly, centrifuging, adding 2 percent CaCl2 solution with the volume of 10-15 times that of the obtained algae powder, performing ultrasonic extraction at the temperature of 60-70 ℃, stirring by using a high-shear emulsifying machine at the rotation speed of 13000 plus 19000rpm for 10 minutes, stopping for 10 minutes, closing the ultrasonic after 10-20 minutes, continuing extracting for 60-120 minutes, extracting for 2 times, separating and combining clear liquid; fourthly, removing brown algae starch, respectively carrying out ultrafiltration on the extracting solution under the conditions that the molecular weight cut-off is 5k, 10k and 100kDa, respectively collecting 200mL of permeate, concentrating the permeate to 10mL under reduced pressure, determining the content of fucoidan by adopting a methylene blue method, selecting the highest molecular weight cut-off which does not detect the fucoidan for ultrafiltration so as to ensure that the brown algae starch is removed to the maximum extent, and continuously carrying out ultrafiltration concentration to 1/10-1/15 of the original volume; and fifthly, adding 3-4 times volume of 95% ethanol for precipitation, drying the precipitate at 50-60 ℃ to obtain polysaccharide, measuring the monosaccharide composition of the polysaccharide by using a PMP-pre-column derivatization high performance liquid chromatography, and measuring the sulfate group content of the polysaccharide by using a barium chloride-gelatin turbidimetry method to determine that the obtained polysaccharide is fucoidan.
In the third step, a combined extraction mode of hot CaCl2 solution-ultrasonic wave-high shear emulsifying machine is adopted for extraction.
In the combined extraction method of fucoidan, after the third step of ultrasonic extraction is carried out for 10-20 minutes, the ultrasonic wave is turned off, and the extraction is continued for 60-120 minutes.
The fourth step is removing the algin by ultrafiltration, ultrafiltering the extracting solution under the conditions of the molecular weight cut-off of 5k, 10k and 100kDa respectively, collecting 200mL of permeate liquid respectively, concentrating the permeate liquid to 10mL by decompression, determining the content of fucosan sulfate by adopting a methylene blue method, selecting the highest molecular weight cut-off which does not detect the fucosan sulfate, and carrying out ultrafiltration to remove the algin.
Advantageous effects
1. The invention adopts hot CaCl2The combined mode of solution-ultrasonic wave-high shear emulsifying machine is adopted for extraction, the operation is simple, and the brown algae isThe yield of the sugar gum is obviously higher than that of common hot CaCl under the same condition2The extraction rate of the solution.
The fucoidan generally contains algin, algin and fucoidan, and the fucoidan usually contains the algin in the conventional extraction.
According to the invention, the molecular weight cut-off is selected according to the content of fucoidan sulfate in the permeate, so that the fucoidan starch of different brown algae can be more accurately removed, the loss of fucoidan can be reduced, and the purity of the fucoidan can be improved.
Drawings
FIG. 1 Sargassum fusiforme polysaccharide monosaccharide composition.
FIG. 2 the composition of sargassum horneri polysaccharide monosaccharides.
Detailed Description
Example 1
A combined extraction method of fucoidan comprises five steps, firstly, collecting brown algae, oven drying at 40-50 deg.C, pulverizing with high-speed multifunctional pulverizer, and sieving with 60-80 mesh sieve; the second step is to add 95 percent ethanol with 10 to 15 times volume for three times of degreasing, and each time lasts for about 480 and 720 minutes; thirdly, centrifuging, adding 2 percent CaCl2 solution with the volume of 10-15 times that of the obtained algae powder, performing ultrasonic extraction at the temperature of 60-70 ℃, stirring by using a high-shear emulsifying machine at the rotation speed of 13000 plus 19000rpm for 10 minutes, stopping for 10 minutes, closing the ultrasonic after 10-20 minutes, continuing extracting for 60-120 minutes, extracting for 2 times, separating and combining clear liquid; fourthly, removing brown algae starch, respectively carrying out ultrafiltration on the extracting solution under the conditions that the molecular weight cut-off is 5k, 10k and 100kDa, respectively collecting 200mL of permeate, concentrating the permeate to 10mL under reduced pressure, determining the content of fucoidan by adopting a methylene blue method, selecting the highest molecular weight cut-off which does not detect the fucoidan for ultrafiltration so as to ensure that the brown algae starch is removed to the maximum extent, and continuously carrying out ultrafiltration concentration to 1/10-1/15 of the original volume; and fifthly, adding 3-4 times volume of 95% ethanol for precipitation, drying the precipitate at 50-60 ℃ to obtain polysaccharide, measuring the monosaccharide composition of the polysaccharide by using a PMP-pre-column derivatization high performance liquid chromatography, and measuring the sulfate group content of the polysaccharide by using a barium chloride-gelatin turbidimetry method to determine that the obtained polysaccharide is fucoidan.
Example 2
The combined extraction method of fucoidan described in example 1, wherein hot CaCl is used as the third step2The extraction is carried out by combining a solution-ultrasonic wave-high shear emulsifying machine.
Example 3
The combined extraction method of fucoidan described in example 1, wherein the third step of ultrasonic extraction is performed for 10-20 minutes, then the ultrasonic extraction is turned off, and the extraction is continued for 60-120 minutes.
Example 4
In the combined extraction method of fucoidan described in embodiment 1, the fourth step is to remove the fucoidan by ultrafiltration, and select the cut-off molecular weight according to the fucoidan content in the permeate, so that the fucoidan of different algin can be removed more accurately, and the loss of fucoidan can be reduced.
Example 5
The combined extraction method of fucoidan described in example 1 is to take 500g of Hizikia fusiforme, dry the Hizikia fusiforme at 40-50 ℃, pulverize the Hizikia fusiforme by a high-speed multifunctional pulverizer, and sieve the Hizikia fusiforme with a 60-80 mesh sieve. Defatting with 10-15 times volume of 95% ethanol for three times (each time for 8-12 hr), and centrifuging. Adding 10-15 times of 2% CaCl into the obtained algae powder2Ultrasonic extracting the solution at 60-70 deg.C, stirring with high-shear emulsifying machine at 13000-19000rpm for 10 min, stopping ultrasonic extracting after 10-20 min, extracting for 1-2 hr for 2 times, separating and combining the clear liquid. Respectively ultrafiltering the extractive solution under the conditions of molecular weight cut-off of 5k, 10k and 100kDa, respectively collecting 200mL of permeate, concentrating under reduced pressure to 10mL, determining fucoidan sulfate content by using a methylene blue method, and selecting the highest molecular weight cut-off of which fucoidan sulfate is not detected for ultrafiltration to ensure that the brown algae starch is removed to the maximum extent. According to the determination result, selecting 10kDa to continue ultrafiltration, concentrating to 1/10-1/15 of the original volume, adding 3-4 times of 95% ethanol for precipitation, drying the precipitate at 50 deg.C to obtain Sargassum fusiforme polysaccharide with yield of 5.29%, which is higher than that obtained by only using hot CaCl under the same condition2The yield in the solution extraction was 3.15%. The monosaccharide composition determination shows that the sargassum fusiforme is richThe sugar mainly comprises mannose, glucosamine, glucuronic acid, glucose, galactose, xylose and fucose, the molar ratio (mol%) is respectively 8.17%, 2.74%, 2.01%, 1.45%, 25.56%, 0.24% and 59.83%, the fucose content is higher, and the glucose content is extremely low. Barium chloride-gelatin turbidimetry showed that the sulfate group content of the polysaccharide was 21.23%. Therefore, the obtained sargassum fusiforme polysaccharide is fucoidan sulfate, namely fucoidan.
Example 6
The combined extraction method of fucoidan described in example 1 is to take 500g of fucoidan, dry the fucoidan at 40-50 ℃, pulverize the fucoidan by a high-speed multifunctional pulverizer, and pass through a 60-80 mesh sieve. Defatting with 10-15 times volume of 95% ethanol for three times (each time for 8-12 hr), and centrifuging. Adding 10-15 times of 2% CaCl into the obtained algae powder2Ultrasonic extracting the solution at 60-70 deg.C, stirring with high-shear emulsifying machine at 13000-19000rpm for 10 min, stopping ultrasonic extracting after 10-20 min, extracting for 1-2 hr for 2 times, separating and combining the clear liquid. Respectively ultrafiltering the extractive solution under the conditions of molecular weight cut-off of 5k, 10k and 100kDa, respectively collecting 200mL of permeate, concentrating under reduced pressure to 10mL, determining fucoidan sulfate content by using a methylene blue method, and selecting the highest molecular weight cut-off of which fucoidan sulfate is not detected for ultrafiltration to ensure that the brown algae starch is removed to the maximum extent. According to the determination result, selecting 100kDa to continue ultrafiltration, concentrating to 1/10-1/15 of the original volume, adding 3-4 times of 95% ethanol for precipitation, drying the precipitate at 50 ℃ to obtain Sargassum horneri polysaccharide with yield of 5.78%, which is higher than that obtained by only using hot CaCl under the same condition2The yield in the solution extraction was 3.26%. The monosaccharide composition determination result shows that the sargassum horneri polysaccharide mainly comprises mannose, glucuronic acid, galactose, xylose and fucose, the molar ratio (moL%) is 14.79%, 11.98%, 30.26%, 5.31% and 37.66%, respectively, the fucose content is high, and the sargassum horneri polysaccharide does not contain glucose. Barium chloride-gelatin turbidimetry showed that the sulfate group content of the polysaccharide was 17.22%. Therefore, the obtained sargassum horneri polysaccharide is fucoidan sulfate, i.e. fucoidan.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (4)
1. A combined extraction method of fucoidan is characterized in that the method comprises five steps, wherein brown algae is taken in the first step, dried at 40-50 ℃, crushed by a high-speed multifunctional crusher and sieved by a sieve with 60-80 meshes; the second step is to add 95 percent ethanol with 10 to 15 times volume for three times of degreasing, and each time lasts for about 480 and 720 minutes; thirdly, centrifuging, adding 2 percent CaCl2 solution with the volume of 10-15 times that of the obtained algae powder, performing ultrasonic extraction at the temperature of 60-70 ℃, stirring by using a high-shear emulsifying machine at the rotation speed of 13000 plus 19000rpm for 10 minutes, stopping for 10 minutes, closing the ultrasonic after 10-20 minutes, continuing extracting for 60-120 minutes, extracting for 2 times, separating and combining clear liquid; fourthly, removing brown algae starch, respectively carrying out ultrafiltration on the extracting solution under the conditions that the molecular weight cut-off is 5k, 10k and 100kDa, respectively collecting 200mL of permeate, concentrating the permeate to 10mL under reduced pressure, determining the content of fucoidan by adopting a methylene blue method, selecting the highest molecular weight cut-off which does not detect the fucoidan for ultrafiltration so as to ensure that the brown algae starch is removed to the maximum extent, and continuously carrying out ultrafiltration concentration to 1/10-1/15 of the original volume; and fifthly, adding 3-4 times volume of 95% ethanol for precipitation, drying the precipitate at 50-60 ℃ to obtain polysaccharide, measuring the monosaccharide composition of the polysaccharide by using a PMP-pre-column derivatization high performance liquid chromatography, and measuring the sulfate group content of the polysaccharide by using a barium chloride-gelatin turbidimetry method to determine that the obtained polysaccharide is fucoidan.
2. The combined extraction method of fucoidan according to claim 1, wherein the third step is performed by a combination of hot CaCl2 solution-ultrasonic wave-high shear emulsifying machine.
3. The combined extraction method of fucoidan according to claim 1, wherein the third step of ultrasonic extraction is performed for 10-20 minutes, and then the ultrasonic extraction is stopped for 60-120 minutes.
4. The combined extraction method of fucoidan according to claim 1, wherein the fourth step is to remove the fucoidan by ultrafiltration, to ultrafilter the extractive solutions at molecular weight cut-offs of 5k, 10k, and 100kDa, to collect 200mL of the permeate, to concentrate the permeate under reduced pressure to 10mL, to determine the fucoidan content by methylene blue method, to select the highest molecular weight cut-off that the fucoidan is not detected, to ultrafiltrate and to remove the fucoidan.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112500504A (en) * | 2020-12-11 | 2021-03-16 | 山东省科学院生物研究所 | Tumor targeting fucosan sulfate and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112500504A (en) * | 2020-12-11 | 2021-03-16 | 山东省科学院生物研究所 | Tumor targeting fucosan sulfate and preparation method thereof |
| CN112500504B (en) * | 2020-12-11 | 2022-07-29 | 山东省科学院生物研究所 | Tumor targeting fucosan sulfate and preparation method thereof |
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Application publication date: 20200515 |