CN116375901A - Preparation method of asparagus fucoidin, obtained fucoidin and application of fucoidin - Google Patents
Preparation method of asparagus fucoidin, obtained fucoidin and application of fucoidin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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Abstract
The invention provides a preparation method of asparagus fucoidin, the obtained fucoidin and application thereof, and relates to the technical field of food processing, wherein the preparation method comprises the following steps: 1) Drying thallus Gracilariae, and pulverizing; 2) Mixing Gracilaria verrucosa powder with ethanol, refluxing for decolorizing, collecting residue, and drying; 3) Mixing decolorized Gracilaria verrucosa powder with water, treating in water bath, centrifuging, collecting supernatant, concentrating by rotary evaporation, mixing with ethanol, standing, precipitating, redissolving, and vacuum freeze drying; 4) Mixing the crude extract with water, centrifuging, collecting supernatant, eluting with anion exchange column, dialyzing, loading onto endotoxin removal column, collecting effluent, and vacuum freeze drying to obtain purified Gracilaria lemaneiformis fucoidin. The preparation method has the advantage of higher purity, the total sugar content can reach 97.82%, the antiallergic activity of the obtained fucoidin purified end product is obviously improved, and the in-vitro inhibition activity of the hyaluronidase can reach 57.39%.
Description
Technical Field
The invention relates to the technical field of food processing, in particular to a preparation method of asparagus fucoidin, the obtained fucoidin and application thereof.
Background
With the increasing population of global food allergy, food allergy has become an important food safety problem that both endangers the health of the people and affects the food processing industry. At present, about 2.5% of the population worldwide has food allergy, and the figure is gradually rising, however, no method for radically treating food allergy exists in medicine to date. In order to control the increasing trend of food allergy, the development of natural active substances having antiallergic activity for preventing and controlling food allergy has attracted a great deal of attention from students in the home and abroad. Research shows that fucoidin has biological activities such as anti-tumor, antioxidation and the like, however, the research on the antiallergic effect of fucoidin is still fresh and reported at present, so that an extraction and purification method of high-purity fucoidin is constructed, and the activity of the fucoidin for resisting food anaphylactic reaction in vitro and in vivo is further explored, which has important significance for developing antiallergic functional foods and novel antiallergic drugs.
The asparagus (Gracilaria lemaneiformis) is a common food material, is a temperate seaweed belonging to the Gracilaria family of the red alga, is planted in a large amount in coastal areas such as Shandong, guangdong and Fujian, has increased yield year by year in recent years, and is mainly used as a raw material for producing food agar and can also be used for developing green food. The asparagus is rich in nutrition and fucoidin, the fucoidin content of the asparagus is about 30% of the dry weight, and the asparagus has high utilization value. At present, few scholars research on a separation and purification method and biological activity of asparagus fucoidin, and research reports on antiallergic activity of asparagus fucoidin are still available.
Disclosure of Invention
The invention aims to provide a preparation method of asparagus fucoidin, the obtained fucoidin and application thereof, so as to solve the problem of how to extract the fucoidin with antiallergic activity from asparagus.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of asparagus fucoidin, which comprises the following steps:
1) Drying thallus Gracilariae, and pulverizing to obtain thallus Gracilariae algae powder;
2) Mixing asparagus algae powder and absolute ethyl alcohol according to the proportion of 1mg to 2-6 mL, carrying out reflux decoloration treatment for 1-3 times, taking filter residues, and drying to obtain decolored asparagus algae powder;
3) The decolorized asparagus algae powder and water are mixed according to 10g: 400-600 mL of the raw materials are mixed, treated in a water bath, centrifuged for the first time, the supernatant is taken out, and concentrated by rotary evaporation to 10-30% of the original volume, thus obtaining concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to the volume ratio of 1:2-6, standing, leaving a precipitate, redissolving, and then performing first vacuum freeze drying to obtain the asparagus fucoidin crude extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing 0.5-1.5 mL, centrifuging, collecting supernatant, eluting with anion exchange column, collecting eluent, dialyzing the eluent to obtain dialysate, loading the dialysate onto a Detoxil-Gel endotoxin removal column, collecting effluent with the volume of the second time to five times of that of the column, and lyophilizing under vacuum to obtain purified Gracilaria verrucosa fucoidin.
Preferably, the temperature of the drying in the step 1) is 50-70 ℃, the drying time is 2-6 hours, and the particle size of the asparagus algae powder obtained by crushing is 40-80 meshes.
Preferably, the temperature of the reflux decolorization treatment in the step 2) is 40-60 ℃, and the time of each reflux decolorization treatment is 1-4 h.
Preferably, the temperature of the drying in the step 2) is 30-50 ℃, and the drying time is 2-6 h.
Preferably, the temperature of the water bath treatment in the step 3) is 90-110 ℃, and the time of the water bath treatment is 2-4 hours; the rotating speed of the first centrifugation is 3000-5000 r/min, and the time of the first centrifugation is 10-30 min.
Preferably, the temperature of the rotary evaporation in the step 3) is 60-80 ℃, the standing time is 8-12 h, the re-dissolving solvent is water, the temperature of the first vacuum freeze drying is-70-90 ℃, and the time of the first vacuum freeze drying is 24-72 h.
Preferably, the rotation speed of the second centrifugation in the step 4) is 7000-9000 r/min, and the time of the second centrifugation is 5-15 min.
Preferably, the dialysis in the step 4) is performed in a dialysis bag, the molecular cutoff amount of the dialysis bag is 3kDa, and the dialysis time is 24-72 hours; the temperature of the second vacuum freeze drying is-70 to-90 ℃, and the time of the second vacuum freeze drying is 24-72 h.
The invention also provides the asparagus fucoidin obtained by the preparation method of the asparagus fucoidin.
The invention also provides asparagus fucoidin obtained by the preparation method or application of the asparagus fucoidin in preparing an antiallergic product.
The invention provides a preparation method of asparagus fucoidin and the obtained fucoidin and application thereof, which comprises the steps of preparing asparagus fucoidin powder, decolorizing with ethanol, leaching with hot water, concentrating under reduced pressure, precipitating with ethanol, eluting with DEAE-Sepharose Fast Flow anion exchange column (eluting with water to remove impurities and 2mol/L NaCl), dialyzing, performing Detoxi-Gel column chromatography (removing lipopolysaccharide endotoxin), and performing vacuum freeze-drying to obtain high-purity asparagus fucoidin, wherein compared with the purified matter of the traditional polysaccharide extraction method, the preparation method has the advantage of higher purity, the total sugar content can reach 97.82%, the content of impurities such as protein, pigment, lipopolysaccharide endotoxin and the like in the purified matter is greatly reduced, the antiallergic activity of the obtained fucoidin purified end product is obviously improved, and the in-vitro inhibitory activity of hyaluronidase can reach 57.39%, and the advantage is obvious; the fucoidin with antiallergic activity is extracted from asparagus, high-valued deep processing utilization of asparagus is realized, and compared with the extraction mode of the existing polysaccharide active substance, the yield and purity of the fucoidin are higher, the antiallergic activity of the fucoidin is stronger, the safety is higher, the fucoidin is a potential source capable of playing a role in the field of antiallergic, technical support is provided for developing food or medicine raw materials with the function of preventing and treating food allergy, and the high-valued utilization and industrial development of asparagus biological resources are actively promoted.
Drawings
FIG. 1 is an evaluation of RBL-2H3 mast cell model of antiallergic activity in vitro of purified asparagus fucoidin prepared in example 1 (A: degranulation; B: histamine secretion; C: IL-4 secretion; D: TNF-. Alpha.secretion);
FIG. 2 is a flow chart of immunization of an allergic mouse animal model;
FIG. 3-A shows the mice allergic symptom scores (PBS negative control group a, TM positive control group b, low dose Gracilaria verrucosa fucoidan group c and high dose Gracilaria verrucosa fucoidan group d) of the in vivo antiallergic activity of the purified Gracilaria verrucosa fucoidan prepared in example 1;
FIG. 3-B shows the levels of histamine in the serum of mice having antiallergic activity in vivo of the purified Gracilaria verrucosa fucoidin prepared in example 1 (PBS negative control group a, TM positive control group B, low dose Gracilaria verrucosa fucoidin group c and high dose Gracilaria verrucosa fucoidin group d);
FIG. 3-C shows IgE antibody levels in the serum of mice with antiallergic activity in vivo of the purified Gracilaria verrucosa fucoidin prepared in example 1 (PBS negative control group a group, TM positive control group b group, low dose Gracilaria verrucosa fucoidin group C group and high dose Gracilaria verrucosa fucoidin group d group);
FIG. 3-D shows the IgG1 antibody levels (PBS negative control group a, TM positive control group b, low dose Gracilaria verrucosa fucoidan group c and high dose Gracilaria verrucosa fucoidan group D) in the mouse serum of the antiallergic activity in vivo of the purified Gracilaria verrucosa fucoidan prepared in example 1.
Detailed Description
The invention provides a preparation method of asparagus fucoidin, which comprises the following steps:
1) Drying thallus Gracilariae, and pulverizing to obtain thallus Gracilariae algae powder;
2) Mixing asparagus algae powder and absolute ethyl alcohol according to the proportion of 1mg to 2-6 mL, carrying out reflux decoloration treatment for 1-3 times, taking filter residues, and drying to obtain decolored asparagus algae powder;
3) The decolorized asparagus algae powder and water are mixed according to 10g: 400-600 mL of the raw materials are mixed, treated in a water bath, centrifuged for the first time, the supernatant is taken out, and concentrated by rotary evaporation to 10-30% of the original volume, thus obtaining concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to the volume ratio of 1:2-6, standing, leaving a precipitate, redissolving, and then performing first vacuum freeze drying to obtain the asparagus fucoidin crude extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing 0.5-1.5 mL, centrifuging, collecting supernatant, eluting with anion exchange column, collecting eluent, dialyzing the eluent to obtain dialysate, loading the dialysate onto a Detoxil-Gel endotoxin removal column, collecting effluent with the volume of the second time to five times of that of the column, and lyophilizing under vacuum to obtain purified Gracilaria verrucosa fucoidin.
In the invention, asparagus is dried and crushed to obtain asparagus algae powder. The temperature of the drying is preferably 50 to 70 ℃, more preferably 52 to 68 ℃, still more preferably 54 to 66 ℃, still more preferably 58 to 64 ℃, the time of the drying is preferably 2 to 6 hours, more preferably 2.5 to 5.5 hours, still more preferably 3 to 5 hours; the particle size of the asparagus algae powder obtained by crushing is preferably 40-80 meshes, more preferably 50-70 meshes, and even more preferably 55-65 meshes; the asparagus algae powder is placed in a sealing bag for sealing and preservation, and is preserved at the temperature of minus 20 ℃ for standby.
In the invention, step 2) asparagus algae powder and absolute ethyl alcohol are mixed according to the proportion of 1mg to 2-6 mL, reflux decolorization treatment is carried out for 1-3 times, filter residues are taken out, and the decolorized asparagus algae powder is obtained after drying; the mixing ratio of the asparagus algae powder to the absolute ethyl alcohol is 1 mg:2-6 mL, more preferably 1 mg:3-5 mL, the temperature of the reflux decolorization treatment is preferably 40-60 ℃, more preferably 45-55 ℃, still more preferably 48-52 ℃, the reflux decolorization treatment is 1-3 times, more preferably 2 times, the time of each reflux decolorization treatment is preferably 1-4 h, more preferably 1.5-3.5 h, still more preferably 2-3 h, and after the reflux decolorization treatment, filter paper is preferably adopted for filtering, filter residues are taken, and pigments and alcohol-soluble impurities are removed; the drying temperature is preferably 30 to 50 ℃, more preferably 35 to 45 ℃, still more preferably 38 to 43 ℃, the drying time is preferably 2 to 6 hours, more preferably 2.5 to 5.5 hours, still more preferably 3 to 5 hours, and the drying purpose is to volatilize ethanol.
In the invention, step 3) decolorized asparagus algae powder and water are mixed according to 10g: 400-600 mL of the raw materials are mixed, treated in a water bath, centrifuged for the first time, the supernatant is taken out, and concentrated by rotary evaporation to 10-30% of the original volume, thus obtaining concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to the volume ratio of 1:2-6, standing, leaving a precipitate, redissolving, and then performing first vacuum freeze drying to obtain the asparagus fucoidin crude extract; the mixing ratio of the decolored asparagus algae powder to the water is 10g:400 to 600mL, more preferably 10g:450 to 550mL, still more preferably 10g: 480-520 mL, wherein the temperature of the water bath treatment is preferably 90-110 ℃, more preferably 95-105 ℃, still more preferably 98-102 ℃, and the time of the water bath treatment is preferably 2-4 h, more preferably 2.5-3.5 h; after the water bath treatment, the water bath treatment is preferably cooled to 20-30 ℃, and then the first centrifugation is carried out, wherein the rotating speed of the first centrifugation is preferably 3000-5000 r/min, more preferably 3500-4500 r/min, still more preferably 3800-4200 r/min, the time of the first centrifugation is preferably 10-30 min, more preferably 15-25 min, still more preferably 18-22 min, the supernatant is filtered by filter paper after the first centrifugation, the temperature of rotary evaporation is preferably 60-80 ℃, more preferably 65-75 ℃, still more preferably 68-72 ℃, and the mixing volume ratio of the concentrated solution and absolute ethyl alcohol is 1:2-6, more preferably 1:2.5-5.5, still more preferably 1:3-5; the standing time is preferably 8-12 hours, more preferably 9-11 hours, still more preferably 9.5-10.5 hours, and the standing temperature is preferably 0-4 ℃, more preferably 1-3 ℃, still more preferably 2 ℃; precipitating with absolute ethanol, and collecting precipitate; the redissolution solvent is preferably water, the temperature of the first vacuum freeze drying is preferably-70 to-90 ℃, more preferably-75 to-85 ℃, still more preferably-78 to-82 ℃, and the time of the first vacuum freeze drying is preferably 24 to 72 hours, more preferably 36 to 60 hours, still more preferably 42 to 54 hours.
In the invention, the step 4) of the crude extract of asparagus fucoidin and water are mixed according to 5mg: mixing 0.5-1.5 mL, centrifuging, collecting supernatant, eluting with DEAE-Sepharose FastFlow anion exchange column, collecting eluate, dialyzing in deionized water, loading dialysate into a Detoxil-Gel endotoxin removal column, collecting effluent with the volume of the second time to five times of that of the column, and lyophilizing under vacuum to obtain purified Gracilaria verrucosa fucoidin; the mixing ratio of the asparagus fucoidin crude extract and water is 5mg:0.5 to 1.5mL, more preferably 5mg:0.6 to 1.4mL, and still more preferably 5mg: the rotation speed of the second centrifugation is preferably 7000-9000 r/min, more preferably 7200-8800 r/min, still more preferably 7400-8600 r/min, and the time of the second centrifugation is preferably 5-15 min, more preferably 6-14 min, still more preferably 8-12 min; the passing through elution is preferably that supernatant is passed through DEAE-Sepharose FastFlow anion exchange column (column type: XK26/100,26 mm. Times.100 cm), one column volume is eluted with deionized water at an elution rate of 1.5mL/min, impurities are removed, one column volume is eluted with NaCl at 2mol/L at an elution rate of 1.5mL/min, and eluent is collected; loading the eluent into a dialysis bag, wherein the dialysis is preferably performed in the dialysis bag, the molecular cut-off of the dialysis bag is preferably 3kDa, the dialysis time is preferably 24-72 h, more preferably 36-60 h, still more preferably 42-54 h, then loading the dialysate into a detox-Gel endotoxin removal column, removing lipopolysaccharide endotoxin in the dialysate, discarding one column volume effluent which is discharged first after loading, and then starting to collect effluent of a second to five times of column volumes; the effluent is subjected to second vacuum freeze-drying, wherein the temperature of the second vacuum freeze-drying is preferably-70 to-90 ℃, more preferably-75 to-85 ℃, still more preferably-78 to-82 ℃, and the time of the second vacuum freeze-drying is preferably 24 to 72 hours, more preferably 36 to 60 hours, still more preferably 42 to 54 hours; obtaining purified asparagus fucoidin, and preserving at-20 ℃ for standby.
The invention also provides the asparagus fucoidin obtained by the preparation method of the asparagus fucoidin.
The invention also provides asparagus fucoidin obtained by the preparation method or application of the asparagus fucoidin in preparing an antiallergic product, wherein the product is one of a medicine and a food.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1) Oven drying thallus Gracilariae at 60deg.C for 4 hr, pulverizing to 60 mesh to obtain thallus Gracilariae powder, sealing in a bag, and preserving at-20deg.C;
2) Mixing Gracilaria verrucosa powder with 4mL of absolute ethyl alcohol according to the proportion of 1mg, refluxing for decoloration treatment, refluxing at 50 ℃ for 3 hours, removing supernatant, taking filter residues, adding 4 times of absolute ethyl alcohol with the volume of 50 ℃ for refluxing for 1 hour, filtering by adopting filter paper, taking the filter residues, and drying at 40 ℃ for 4 hours to obtain decolored Gracilaria verrucosa powder;
3) The decolorized asparagus algae powder and deionized water are mixed according to 10g: mixing at 500mL ratio, treating in water bath at 100deg.C for 3 hr, cooling to 25deg.C, centrifuging at 4000r/min for 20min, filtering with filter paper, collecting supernatant, and concentrating by rotary evaporation at 70deg.C to 20% of original volume to obtain concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to a volume ratio of 1:4, standing at 4 ℃ for 10 hours, leaving a precipitate, re-dissolving with deionized water, and then performing first vacuum freeze drying at-80 ℃ for 48 hours to obtain a crude asparagus fucoidin extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing at a ratio of 1mL, centrifuging at 25deg.C for 10min at 8000r/min, collecting supernatant, subjecting to DEAE-Sepharose FastFlow anion exchange column (column: XK26/100,26mm×100 cm) gradient elution, eluting with deionized water at an elution rate of 1.5mL/min for one column volume, removing impurities, eluting with 2mol/L NaCl at an elution rate of 1.5mL/min for one column volume, collecting eluate, loading into a 3kDa dialysis bag, dialyzing in deionized water for 48 hr, loading the dialysate into a detox-Gel endotoxin removal column, discarding one column volume effluent from the first column after loading, collecting effluent of second to five times of column volume, lyophilizing the effluent in second vacuum at-80deg.C for 48 hr to obtain purified Gracilaria lemaneiformis polysaccharide, and preserving at-20deg.C for use.
Example 2
1) Oven drying thallus Gracilariae at 50deg.C for 2 hr, pulverizing to 40 mesh to obtain thallus Gracilariae powder, sealing in a bag, and preserving at-20deg.C;
2) Mixing Gracilaria verrucosa powder and absolute ethyl alcohol according to the proportion of 1mg to 2mL, carrying out reflux decoloration treatment, carrying out reflux at 40 ℃ for 4 hours, removing supernatant, taking filter residues, adding absolute ethyl alcohol with the volume being 6 times that of the filter residues, carrying out reflux at 60 ℃ for 1 hour, filtering by adopting filter paper, taking the filter residues, and drying at 30 ℃ for 2 hours to obtain decolored Gracilaria verrucosa powder;
3) The decolorized asparagus algae powder and deionized water are mixed according to 10g: mixing 400mL, treating in 90 deg.C water bath for 2h, cooling to 20deg.C, centrifuging at 3000r/min for 15min, filtering with filter paper, collecting supernatant, and concentrating by rotary evaporation at 60deg.C to 10% of original volume to obtain concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to a volume ratio of 1:2, standing at 0 ℃ for 8 hours, reserving sediment, re-dissolving with deionized water, and then performing first vacuum freeze drying at-70 ℃ for 24 hours to obtain a asparagus fucoidin crude extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing at a ratio of 0.5mL, centrifuging at 25deg.C at 7000r/min for 5min, collecting supernatant, subjecting to DEAE-Sepharose FastFlow anion exchange column (column: XK26/100,26mm×100 cm) gradient elution, eluting with deionized water at an elution rate of 1.5mL/min for one column volume, removing impurities, eluting with 2mol/L NaCl at an elution rate of 1.5mL/min for one column volume, collecting eluate, loading into dialysis bag with molecular cutoff of 3kDa, dialyzing in deionized water for 24 hr, loading the dialysate into detoxin-Gel endotoxin removal column, discarding one column volume effluent from the first column after loading, collecting effluent of second to five times column volume, lyophilizing in vacuum at-70deg.C for 24 hr to obtain purified Gracilaria verrucosa polysaccharide, and preserving at-20deg.C for use.
Example 3
1) Oven drying thallus Gracilariae at 70deg.C for 6 hr, pulverizing to 80 mesh to obtain thallus Gracilariae powder, sealing in a bag, and preserving at-20deg.C;
2) Mixing Gracilaria verrucosa powder with anhydrous ethanol according to the ratio of 1mg to 5mL, refluxing for decolorizing, refluxing at 60 ℃ for 2h, removing supernatant, collecting filter residues, adding 3 times of anhydrous ethanol with the volume of 45 ℃ for refluxing for 3h, filtering with filter paper, collecting filter residues, and drying at 45 ℃ for 6h to obtain decolorized Gracilaria verrucosa powder;
3) The decolorized asparagus algae powder and deionized water are mixed according to 10g: mixing 600mL, treating in water bath at 110deg.C for 4 hr, cooling to 30deg.C, centrifuging at 5000r/min for 30min, filtering with filter paper, collecting supernatant, and concentrating by rotary evaporation at 75deg.C to 30% of original volume to obtain concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to a volume ratio of 1:6, standing at 1 ℃ for 12 hours, leaving a precipitate, re-dissolving with deionized water, and then performing first vacuum freeze drying at-90 ℃ for 72 hours to obtain a crude asparagus fucoidin extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing at 1.5mL ratio, centrifuging at 25deg.C for 15min at 9000r/min, collecting supernatant, subjecting to DEAE-Sepharose FastFlow anion exchange column (column: XK26/100,26mm×100 cm) gradient elution, eluting with deionized water at an elution rate of 1.5mL/min for one column volume, removing impurities, eluting with 2mol/L NaCl at an elution rate of 1.5mL/min for one column volume, collecting eluate, loading into 3kDa dialysis bag, dialyzing in deionized water for 72 hr, loading the dialysate into a detox-Gel endotoxin removal column, discarding one column volume effluent from the first column after loading, collecting effluent of second to five times column volume, lyophilizing in vacuum at-90deg.C for 72 hr to obtain purified Gracilaria verrucosa polysaccharide, and preserving at-20deg.C for use.
Example 4
1) Drying thallus Gracilariae at 55deg.C for 3 hr, pulverizing to 50 mesh to obtain thallus Gracilariae powder, sealing in a bag, and preserving at-20deg.C;
2) Mixing asparagus algae powder and absolute ethyl alcohol according to the proportion of 1mg to 3mL, refluxing for decoloration treatment, refluxing at 45 ℃ for 4 hours, removing supernatant, taking filter residues, adding absolute ethyl alcohol with the volume being 5 times that of the filter residues, refluxing at 55 ℃ for 1 hour, taking the filter residues, adding absolute ethyl alcohol with the volume being 4 times that of the filter residues for 50 ℃ for the third time, refluxing for 2 hours, filtering by adopting filter paper, taking the filter residues, and drying at 35 ℃ for 3 hours to obtain decolored asparagus algae powder;
3) The decolorized asparagus algae powder and deionized water are mixed according to 10g:450mL of the raw materials are mixed, treated in a water bath at 100 ℃ for 2.5h, cooled to 25 ℃, subjected to first centrifugation at 3500r/min for 15min, filtered by filter paper, and the supernatant is taken and concentrated to 15% of the original volume by rotary evaporation at 65 ℃ to obtain concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to a volume ratio of 1:3, standing at 2 ℃ for 9 hours, reserving sediment, re-dissolving with deionized water, and then performing first vacuum freeze drying at-75 ℃ for 36 hours to obtain a crude asparagus fucoidin extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing at a ratio of 0.8mL, centrifuging at 25deg.C for 8min at 7500r/min, collecting supernatant, subjecting to DEAE-Sepharose FastFlow anion exchange column (column: XK26/100,26mm×100 cm) gradient elution, eluting with deionized water at an elution rate of 1.5mL/min for one column volume, removing impurities, eluting with 2mol/L NaCl at an elution rate of 1.5mL/min for one column volume, collecting eluate, loading into 3kDa dialysis bag, dialyzing in deionized water for 36 hr, loading the dialysate into a detox-Gel endotoxin removal column, discarding one column volume effluent from the first column after loading, collecting effluent of second to five times column volume, lyophilizing the effluent in vacuum at-75deg.C for 36 hr to obtain purified Gracilaria verrucosa polysaccharide, and preserving at-20deg.C for use.
Example 5
1) Oven drying thallus Gracilariae at 65deg.C for 5 hr, pulverizing to 70 mesh to obtain thallus Gracilariae powder, sealing in a bag, and preserving at-20deg.C;
2) Mixing Gracilaria verrucosa powder with anhydrous ethanol at a ratio of 1mg to 5mL, refluxing for decolorizing, refluxing at 50deg.C for 4 hr, removing supernatant, filtering with filter paper, collecting residue, and drying at 45deg.C for 5 hr to obtain decolorized Gracilaria verrucosa powder;
3) The decolorized asparagus algae powder and deionized water are mixed according to 10g:550mL of the raw materials are mixed, treated for 4 hours in a water bath at 100 ℃, cooled to 25 ℃, centrifuged for 25 minutes at 4500r/min, filtered by filter paper, and the supernatant is taken and concentrated to 20% of the original volume by rotary evaporation at 75 ℃ to obtain concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to a volume ratio of 1:4, standing at 3 ℃ for 11 hours, leaving a precipitate, re-dissolving with deionized water, and then performing first vacuum freeze drying at-85 ℃ for 60 hours to obtain a crude asparagus fucoidin extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing at a ratio of 1.2mL, centrifuging at 25deg.C for 12min at 8500r/min, collecting supernatant, subjecting to DEAE-Sepharose FastFlow anion exchange column (column: XK26/100,26mm×100 cm) gradient elution, eluting with deionized water at an elution rate of 1.5mL/min for removing impurities, eluting with 2mol/L NaCl at an elution rate of 1.5mL/min for removing impurities, collecting eluate, loading into 3kDa dialysis bag, dialyzing in deionized water for 60 hr, loading the dialysate into a detox-Gel endotoxin removal column, discarding one column volume effluent from the first column after loading, collecting effluent of second to five times column volume, lyophilizing in vacuum at-85deg.C for 60 hr to obtain purified Gracilaria lemaneiformis fucoidin, and storing at-20deg.C for standby.
Experimental example 1
Physicochemical property analysis of asparagus fucoidin
Phenol-sulfuric acid process:
the purified asparagus fucoidan prepared in example 1 was used to draw a standard curve using glucose, and 0.1mol/L glucose solutions (10. Mu.L, 20. Mu.L, 40. Mu.L, 80. Mu.L, 160. Mu.L, 320. Mu.L and 500. Mu.L) were each aspirated into a 5mL centrifuge tube, and then made up to 500. Mu.L with ultrapure water, and then 0.3mL of a 6% phenol (w/v) solution was added and mixed well. 1.5mL of concentrated sulfuric acid was added, mixed well, heated in a boiling water bath for 20min, immediately cooled in ice water, and absorbance at 490nm was measured with an ultraviolet-visible photometer. Meanwhile, a standard curve was drawn with 500. Mu.L of ultrapure water as a blank, the absorbance as an ordinate, and the glucose content (. Mu.g) as an abscissa. Sucking 0.5mL of the purified asparagus fucoidin solution prepared in the example 1, measuring the absorbance at 490nm according to the steps, and calculating the fucoidin content in the sample by using a standard curve.
Sulfuric acid-carbazole method:
standard curves were prepared using glucuronic acid, and 100. Mu.g/mL glucuronic acid solution was added to each test tube: 0.10mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL, make up to 1mL with deionized water, and take 1mL of deionized water as a blank. The purified asparagus fucoidin prepared in example 1 was prepared into 1mL of 1mg/mL solution with distilled water. 6mL of concentrated sulfuric acid (98% by mass) is added into a test tube respectively, the mixture is carefully and oscillated and mixed uniformly, the mixture is put into a water bath at 100 ℃ for 20min, the mixture is taken out and cooled, 200 mu L of 0.15% carbazole-absolute ethyl alcohol solution is added, the mixture is placed in a dark place at room temperature for 2h after being fully mixed uniformly, and the absorbance is measured at 526nm wavelength. And drawing a standard curve by taking the concentration (mug/mL) of the standard substance as an abscissa and the light absorption value as an ordinate, and calculating the content of the aldonic acid in the sample.
Gelatin-barium chloride process:
a standard curve was prepared using a potassium sulfate standard, and 600. Mu.g/mL of sulfate solution (converted from 1.0875mg/mL of potassium sulfate solution) was added to each test tube: 20. Mu.L, 40. Mu.L, 80. Mu.L, 120. Mu.L, 160. Mu.L, 200. Mu.L, 1mol/L HCl was used to make up to 200. Mu.L, and 200. Mu.L deionized water was used as a blank. The purified asparagus fucoidin prepared in example 1 was prepared with deionized water to a concentration of 600. Mu.g/mL in 200. Mu.L. 3.8mL of 3% trichloroacetic acid solution, 1mL of 5% gelatin solution, were added to the test tubes, mixed well under shaking, allowed to stand at room temperature for 15min, and absorbance A was measured at a wavelength of 360nm 1 The method comprises the steps of carrying out a first treatment on the surface of the The 5% gelatin solution was replaced with 5% BaCl 2 -5% gelatin reagent, absorbance A measured at 360nm wavelength 2 . Absorbance A on the abscissa of standard concentration (. Mu.g/mL) 2 -A 1 And drawing a standard curve for the ordinate, and calculating the sulfate radical content in the sample.
The method for measuring the hyaluronidase in-vitro inhibition activity comprises the following steps:
four EP tubes were labeled with a (control solution), B (control blank solution), C (fucoidan-like liquid prepared in example 1), and D (fucoidan sample blank liquid prepared in example 1), respectively, followed by the following steps:
TABLE 1 Experimental flow for determining hyaluronidase in vitro inhibitory Activity
The calculation formula of the inhibition percentage is shown as 1:
inhibition ratio = [ (A-B) - (C-D) ]/(A-B). Times.100 formula (1)
Performing gradient dilution on the crude extract stock solution, drawing a fitting curve by taking concentration (mg/mL) as an X axis and inhibition rate (%) as a Y axis, and calculating IC according to a fitting curve equation 50 。
BCA protein concentration kit assay protein content:
pierce BCA protein quantification kit, purchased from Thermo Inc. of America.
As shown in Table 2, the content of total sugar in the purified asparagus fucoidin is 97.82% by using a phenol-sulfuric acid method; the content of the aldonic acid is 11.67 percent by utilizing a sulfuric acid-carbazole method, so that the invention can extract the high-purity fucoidin from asparagus; the content of sulfate radical is 15.53% by using gelatin-barium chloride method; the in-vitro inhibition activity of the hyaluronidase is an important index for evaluating the in-vitro antiallergic activity of the active substance, and the in-vitro inhibition rate of the hyaluronidase is 57.39%, which indicates that the purified asparagus fucoidin prepared in the embodiment 1 of the invention has remarkable in-vitro antiallergic activity; the protein content of the fucoidin purified product is 0.75% by using the BCA protein concentration determination kit, which can prove that the purification method can basically remove the protein and obtain the asparagus fucoidin which contains almost no protein.
TABLE 2 physicochemical Properties of the purified Gracilaria verrucosa fucoidan prepared in example 1
Experimental example 2
Cell model evaluation of in vitro antiallergic activity of asparagus fucoidin
RBL-2H3 mast cell assay:
mu.L of the cell suspension (1X 10) was added to each well of a 96-well cell culture plate 6 cells/mL) in a cell incubator (37 ℃,5% CO 2 ) Culturing for 24h, then adding 100ng/mL of anti-DNP-IgE,cell incubator (37 ℃,5% CO) 2 ) Incubate overnight for 12h.
Subsequently, the wells were washed 3 times with PBS, and the purified Gracilaria verrucosa fucoidin prepared in example 1 was dissolved in 100. Mu.L of Tyrode's buffer, and added to the plates at various concentrations (20, 50, 100, 150, 200. Mu.g/mL), respectively, and incubated for 1h with 100. Mu.LPBS in PBS negative control wells.
Subsequently, the content of histamine in the cell culture supernatants was determined using a histamine determination kit (IBL, germany), and the content of IL-4 and TNF-alpha in the cell culture supernatants was determined using an IL-4 and TNF-alpha determination kit (R & D, USA).
The method for detecting the degranulation efficiency of the RBL-2H3 mast cells comprises the following steps: after stimulating RBL-2H3 mast cells with 250ng/mL DNP-BS A for 6H, the cell culture supernatant was recovered and cells were lysed by adding 100. Mu.L Tyrode's buffer (containing 0.1% Triton X-100); finally, respectively adding 25 mu L of supernatant or cell lysate into a 96-well plate, adding 100 mu L of 1.2mM 4-methyl-umbelliferie-ryl-N-acetyl-b-D-glucosamide reagent into each well, reacting for 30min (37 ℃), reading fluorescence value emitted by each well at 360nm and 450nm by using an enzyme-labeled instrument, and calculating the particle removal efficiency (formula 2);
efficiency of degranulation = fluorescence value of supernatant/(fluorescence value of supernatant + fluorescence value of cell lysate) ×100% formula (2).
The above measurement experiments were equally divided into 4 groups, a being PBS, b being tropomyosin, c being low dose Gracilaria verrucosa fucoidin (100 μg/mL), d being high dose Gracilaria verrucosa fucoidin (200 μg/mL).
Mast cells are important effector cells in the allergic reaction process, in-vitro research of allergic reaction often uses RBL-2H3 cells as a mast cell model, and detection of degranulation efficiency of the RBL-2H3 mast cells, secretion levels of histamine, secretion levels of allergic mediators such as allergic cytokines (IL-4 and TNF-alpha) and the like is an important index for evaluating the allergic reaction intensity of the RBL-2H3 mast cell model.
According to the invention, an IgE-mediated RBL-2H3 mast cell model is utilized to verify the in-vitro antiallergic activity of the purified asparagus fucoidin prepared in the example 1, as shown in figure 1, the purified asparagus fucoidin prepared in the example 1 can remarkably inhibit the degranulation efficiency of RBL-2H3 mast cells, and compared with the degranulation efficiency of tropomyosin group cells, the inhibition rates of low-dose asparagus fucoidin (100 mug/mL) and high-dose asparagus fucoidin (200 mug/mL) on the degranulation efficiency of RBL-2H3 mast cells are 31.04% and 53.12%, respectively; the asparagus fucoidin has remarkable inhibition effect on the histamine secretion of RBL-2H3 mast cells, and compared with the histamine secretion amount of tropomyosin group cells, the inhibition rates of low-dose asparagus fucoidin (100 mug/mL) and high-dose asparagus fucoidin (200 mug/mL) on the histamine secretion are 22.51 percent and 38.08 percent respectively; in addition, compared with the secretion amount of the tropomyosin group cell IL-4, the inhibition rate of the low-dose asparagus fucoidin (100 mu g/mL) and the high-dose asparagus fucoidin (200 mu g/mL) on the secretion of the IL-4 cytokines is 22.51 percent and 38.08 percent respectively; in addition, compared with the secretion level of TNF-alpha by tropomyosin group cells, the inhibition rates of the low-dose asparagus fucoidin (100 mug/mL) and the high-dose asparagus fucoidin (200 mug/mL) on the secretion of TNF-alpha cytokines are 26.09% and 43.04%, respectively. Therefore, the purified asparagus fucoidin extracted by the preparation method has remarkable inhibition effect on the allergic reaction of the RBL-2H3 mast cells, and the in-vitro antiallergic activity of the purified asparagus fucoidin prepared by the preparation method is high.
Experimental example 3
Evaluation of in vivo antiallergic Activity of Gracilaria verrucosa fucoidin (purified Gracilaria verrucosa fucoidin prepared in example 1) in mouse animal model
Female BALB/c mice of 6 weeks are taken as study subjects, and the mice are sensitized by taking Tropomyosin (TM) of white prawn as allergen, and an allergic mouse animal model is established, and the concrete scheme is shown in figure 2: all mice were divided into PBS negative control group (group a), TM positive control group (group b), low dose asparagus fucoidin group (group c) and high dose asparagus fucoidin group (group d), 6 mice per group. The TM positive control group and the two groups of asparagus fucoidin groups of mice were respectively injected intraperitoneally with the TM+aluminum hydroxide adjuvant mixture (TM 0.1 mg/mouse) on days 0, 7, 14, 21 of the culture period, and the PBS negative control group of mice was injected with the PBS+aluminum hydroxide adjuvant mixture. Each of the mice of the TM positive control group, the low-dose asparagus fucoidin group and the high-dose asparagus fucoidin group was perfused with 5mg TM, respectively, on days 28 and 35. From day 27 to day 35, all mice were gavaged daily, each mouse was gavaged with 0.2mL of PBS solution, the low dose asparagus fucoidin group was gavaged daily with 0.2mL of PBS solution containing 1mg of asparagus fucoidin, and the high dose asparagus fucoidin group was gavaged daily with 0.2mL of PBS solution containing 5mg of asparagus fucoidin. After 1h from each intraperitoneal injection or lavage, each group of mice was observed for symptoms of allergic reactions and scored. On day 36, all mice were subjected to eyeball-removal blood collection, and serum was collected after centrifugation, and the content of histamine in the mouse serum was analyzed by using a histamine detection kit (IBL company, germany), and the content of IgE antibodies and IgG1 antibodies in the mouse serum was detected by using an enzyme-linked immunosorbent assay (ELISA).
Detection of TM-specific IgE and IgG1 levels in mouse serum: the levels of TM-specific immunoglobulins in the mouse serum were determined by ELISA. 100. Mu.g/mL of TM coated in an ELISA plate, 100. Mu.L/well, 4℃for 16h; TBST washes the plate five times, adds sealing liquid, 300 mu L/hole, seals at 37 ℃ for 2 hours; during this period, the mouse serum samples (1% skim milk) were diluted 1:5, and after blocking, the plates were washed three times with TBST, diluted serum samples were added 100. Mu.L/well, and left at 4℃for 16 hours. The next day, the secondary antibody was diluted with 1% skimmed milk (1:1000 dilution IgE,1:5000 dilution IgG 1), washed five times with TBST, and then 100. Mu.L of the diluted secondary antibody was added, and the mixture was left in an oven at 37℃for 2 hours; washing the plate with TBST for five times after the secondary antibody incubation is finished, adding 100 mu LTMB for color development, and placing in a 37 ℃ oven for color development for 20min; finally all wells were filled with stop solution (2 mol/L H) 2 SO 4 ) The reaction was stopped at 50. Mu.L/well and absorbance at 450nm was measured.
The allergic reaction symptoms of mice are important indicators for evaluating the severity of food allergy in mice. As shown in fig. 3-a, the present invention found that the allergic reaction symptoms of both the gavage low dose fucoidin group mice (1 mg/mouse) and the high dose fucoidin group mice (5 mg/mouse) were significantly relieved, and the degree of alleviation of allergic reaction symptoms was significantly better in the high dose group mice than in the low dose group mice, compared to the tropomyosin positive control group mice.
In addition, as shown in fig. 3-B, by analyzing the histamine secretion level in the serum of the mice, compared with the mice of the tropomyosin positive control group, the intragastric low-dose asparagus fucoidan and the high-dose asparagus fucoidan can both remarkably reduce the content of histamine in the serum of the mice, and the inhibition effect of the intragastric high-dose asparagus fucoidan on histamine secretion is more remarkable.
In addition, as shown in fig. 3-C and 3-D, it can be seen by analyzing the antibody secretion level in the serum of the mice, compared with the tropomyosin positive control mice, the low-dose and high-dose asparagus fucoidin can significantly reduce the content of IgE antibodies and IgG1 antibodies in the serum of the mice, and the inhibition effect of the high-dose asparagus fucoidin on the IgE antibodies and IgG1 antibodies is more significant.
The results show that the asparagus fucoidin can remarkably relieve the anaphylactic reaction symptom of the shrimp tropomyosin allergen sensitized mice, inhibit the secretion of histamine, igE antibodies and IgG1 antibodies in the serum of the mice, and the inhibition degree of the histamine and the antibody secretion is remarkably positively correlated with the gastric lavage dosage of the asparagus fucoidin.
Therefore, based on the analysis of the in vitro inhibition activity of the hyaluronidase of the asparagus fucoidin, the cell model evaluation of the in vitro antiallergic activity of the asparagus fucoidin and the mouse animal model evaluation of the in vivo antiallergic activity of the asparagus fucoidin, the asparagus fucoidin obtained by extraction and purification in the embodiment 1 of the invention has high purity and obvious in vitro and in vivo antiallergic activity, can provide technical support for developing asparagus foods or antiallergic drug raw materials with the function of preventing and treating food allergy, and has important value for promoting the high-value utilization of asparagus biological resources and the industrial development.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of asparagus fucoidin is characterized by comprising the following steps:
1) Drying thallus Gracilariae, and pulverizing to obtain thallus Gracilariae algae powder;
2) Mixing asparagus algae powder and absolute ethyl alcohol according to the proportion of 1mg to 2-6 mL, carrying out reflux decoloration treatment for 1-3 times, taking filter residues, and drying to obtain decolored asparagus algae powder;
3) The decolorized asparagus algae powder and water are mixed according to 10g: 400-600 mL of the raw materials are mixed, treated in a water bath, centrifuged for the first time, the supernatant is taken out, and concentrated by rotary evaporation to 10-30% of the original volume, thus obtaining concentrated solution; mixing the concentrated solution with absolute ethyl alcohol according to the volume ratio of 1:2-6, standing, leaving a precipitate, redissolving, and then performing first vacuum freeze drying to obtain the asparagus fucoidin crude extract;
4) The asparagus fucoidin crude extract and water are mixed according to 5mg: mixing 0.5-1.5 mL, centrifuging, collecting supernatant, eluting with anion exchange column, collecting eluent, dialyzing the eluent to obtain dialysate, loading the dialysate onto a Detoxil-Gel endotoxin removal column, collecting effluent with the volume of the second time to five times of that of the column, and lyophilizing under vacuum to obtain purified Gracilaria verrucosa fucoidin.
2. The method for preparing asparagus fucoidin according to claim 1, wherein the temperature of the drying in the step 1) is 50-70 ℃, the drying time is 2-6 hours, and the particle size of the asparagus fucoidin obtained by crushing is 40-80 meshes.
3. The method for preparing asparagus fucoidin according to claim 1, wherein the temperature of the reflux decolorization treatment in the step 2) is 40-60 ℃, and the time of each reflux decolorization treatment is 1-4 h.
4. The method for preparing asparagus fucoidin according to claim 1, wherein the drying temperature in the step 2) is 30-50 ℃, and the drying time is 2-6 h.
5. The method for preparing asparagus fucoidin according to claim 1, wherein the temperature of the water bath treatment in the step 3) is 90-110 ℃, and the time of the water bath treatment is 2-4 hours; the rotating speed of the first centrifugation is 3000-5000 r/min, and the time of the first centrifugation is 10-30 min.
6. The method for preparing asparagus fucoidin according to claim 1, wherein the temperature of the rotary evaporation in the step 3) is 60-80 ℃, the standing time is 8-12 h, the re-dissolving solvent is water, the temperature of the first vacuum freeze drying is-70-90 ℃, and the time of the first vacuum freeze drying is 24-72 h.
7. The method for preparing asparagus fucoidin according to claim 1, wherein the rotating speed of the second centrifugation in the step 4) is 7000-9000 r/min, and the time of the second centrifugation is 5-15 min.
8. The method for preparing asparagus fucoidin according to claim 1, wherein the dialysis in the step 4) is performed in a dialysis bag, the molecular retention amount of the dialysis bag is 3kDa, and the dialysis time is 24-72 hours; the temperature of the second vacuum freeze drying is-70 to-90 ℃, and the time of the second vacuum freeze drying is 24-72 h.
9. The asparagus fucoidin obtained by the preparation method of asparagus fucoidin according to any one of claims 1 to 8.
10. Use of asparagus fucoidin obtained by the preparation method according to any one of claims 1 to 8 or the asparagus fucoidin according to claim 9 in preparing an antiallergic product.
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