CN103467616A - Preparation method of antiallergic porphyra polysaccharide - Google Patents
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Abstract
The invention discloses a preparation method of antiallergic porphyra polysaccharide, relates to porphyra polysaccharide, and provides a preparation method of the antiallergic porphyra polysaccharide, which is simple in process and strong in operability. The method comprises the following steps: 1) baking porphyra and crushing into powder; 2) dissolving the porphyra powder obtained in the step 1) into water, extracting in water bath at 80-100 DEG C, crushing tissues, centrifuging and obtaining supernatant; 3) concentrating water extract obtained in the step 2), adding ethanol to precipitate, re-dissolving sediments into water after washing, sampling to an anion exchange chromatographic column, determining the sugar content of eluent, and collecting the eluent of which A630 is greater than 0.5; 4) dialyzing the eluent collected in the step 3) by distilled water, freezing and drying in vacuum, so as to obtain the antiallergic porphyra polysaccharide.
Description
Technical field
The present invention relates to a kind of laver amylose, especially relate to a kind of preparation method of antianaphylaxis laver amylose.
Background technology
Food anaphylaxis is the adverse immune response that irritated crowd produces some food proteins, usually can cause series of symptoms, such as asthma, urticaria, diarrhoea etc., has a strong impact on irritated crowd's quality of life, even threat to life safety.U.S.'s epidemiology survey shows, in western countries, there are 8% children and the grownup of 3%-4% to suffer from the food allergy disease, and morbidity is (Scott HS in rising trend still, et al.Food allergy[J] .The Journal of Allergy and Clinical Immunology.2010,125 (2): 116-125).China epidemiology survey shows, infant's food anaphylaxis recall rate be about 7.0%～9.2% (Chen Jing. infant's food anaphylaxis epidemiology survey [D]. Medical University Of Chongqing .2010,2-3).When irritated, the immunologic function of allergy patient's helper T cell can transform to the Th2 type, thereby secrete more Th2 cytokines interleukin-4 (Interleukin4, IL-4), and cause serum immune globulin E (Immune globulin E, IgE) rising (Morita R, et al.Human blood CXCR5+CD4+T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion[J] .Immunity.2011, 34:108 – 121).
Food anaphylaxis has become important public health problem, day by day comes into one's own and pays close attention to, and especially the progress of modernized society is accompanied by the change of diet, is considered to close with the food allergy disease relationship increased gradually in recent years.The food anaphylaxis symptom can not be fundamentally eliminated in traditional pharmacological agent, and can produce resistance and side effect.Result of study shows both at home and abroad, and a large amount of frying oils or the picked-up of the diet of improper lipid acid, may be to affect immunomodulatory to be inclined to anaphylactoid important factor, and the picked-up of seaweeds and vegetables and fruits based food contributes to improve anaphylaxis.Chinese Traditional Medicine ancient books and records and scientific literature are also put down in writing marine alga and are had the effect of improving immunity and digestion.Therefore, extract natural antiallergic activity composition from seaweeds food, study it to immune regulating effect, and develop novel antianaphylaxis protective foods or medicine source goods, will become the inexorable trend of food allergy disease prevention and treatment.
Laver all has a large amount of distributions in the southern and northern marine site of China, for China important propagate economical alga artificially, at present except as common marine food, still produce the raw material of the chemical industry such as agar-agar, medicine.Polysaccharide is the chief component composition of laver, has multiple biological activity and pharmaceutical use, becomes gradually the study hotspot of marine organisms the world of medicine.The laver amylose extracted in foreign literature report nori at present has effect (the Ishihara K that suppresses contact hypersensitivity, et al.Inhibitory effect of porphyran, prepared from dried " Nori ", on contact hypersensitivity in mice[J] .Bioscience, biotechnology, and biochemistry.2005,69 (10): 1824-1830); Alginic acid has effect (the Jeong HJ of antianaphylaxis and inflammation-inhibiting cytokine, et al.Alginic acid has anti-anaphylactic effects and inhibits inflammatory cytokine expression via suppression of nuclear factor-κ B activation[J] .Clinical and Experimental Allergy.2006,36 (6): 785-794); Fucosan in brown alga can suppress anaphylaxis (the Yanase Y that ovalbumin causes, et al.Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization[J] .Biochemical and biophysical research communications.2009,387 (3): 435-439).The domestic literature report, laver amylose has anti-oxidant (Qian Xiaojie, Deng. the extraction of phycobiliprotein and anti-oxidant activity research thereof in porphyra haitanensis. Chinese Journal of Marine Drugs .2008, 27 (2): 42-45), antifatigue (Tingting ZHAO, Deng. the preparation of different molecular weight Porphyra haitanensis polysaccharide and activity of fighting against senium research. Beijing: the .2007:51-54 of the Institute of Oceanology of the Chinese Academy of Sciences) and immunomodulatory (Li Shengjun. the immunoregulation effect of laver amylose to immunosuppressive mice. the .2005:14-30 of Chinese Medical Sciences University) Beijing: the effect such as, so laver amylose has multiple significant biological activity, and rarely have report about the effect of laver amylose antiallergic activity aspect, exploitation antianaphylaxis laver amylose is for taking full advantage of oceanic resources, development functionality food etc. are significant.
Summary of the invention
The object of the present invention is to provide that technique is simple, the preparation method of a kind of antianaphylaxis laver amylose of strong operability.
Concrete steps of the present invention are as follows:
1) laver is dried, then be ground into powder;
2) laver powder step 1) obtained is soluble in water, 80～100 ℃ of water-baths, extracts, and tissue mashing, centrifugal, obtain supernatant;
3) by step 2) the water extraction liquid that obtains is concentrated, adds the ethanol precipitation, by multiple soluble in water after the throw out washing, is splined on the anion-exchange chromatography post, measures the sugar degree of elutriant, collects A630 > 0.5 elutriant;
4) elutriant of step 3) being collected is through distill water dialysis, and vacuum lyophilization, obtain the antianaphylaxis laver amylose.
In step 1), described laver can adopt edible red algae laver; The temperature of described oven dry can be 40～50 ℃.
In step 2) in, the proportioning of described laver powder and water can be 1: (30～50), wherein the laver powder is calculated in mass, and water is calculated by volume; The time that described water-bath is extracted can be 3～5h, and the time of tissue mashing can be 3～5min; Described water can adopt distilled water.
In step 3), described concentrating can adopt Rotary Evaporators by 25%～50% of water extraction liquid simmer down to original volume, and the described condition that adds ethanol to precipitate is 95% ethanol that adds 2～4 times of volumes, and described washing can adopt absolute ethanol washing precipitation 2～4 times; Described water can adopt distilled water.
In step 4), the time of described dialysis can be 48～72h.
The present invention be take edible laver as starting material, by water extraction, tissue mashing extract, the methods such as low pressure is concentrated, alcohol precipitation, the eccysis of ion column stream are assorted, dialysis, prepare the high-purity laver polysaccharide with antiallergic activity.
With existing laver amylose preparation method, compare, the present invention has following outstanding advantages:
In whole preparation process of the present invention, all operations can carry out at normal temperatures, and the laver amylose purity to 75% prepared～80% confirms to have anti-food anaphylaxis activity through experiment.
The accompanying drawing explanation
Fig. 1 is laver amylose DEAE-cellulose52 column chromatography purification collection of illustrative plates in the embodiment of the present invention 1.
Fig. 2 is laver amylose DEAE-cellulose52 column chromatography purification collection of illustrative plates in the embodiment of the present invention 2.
The laver amylose that Fig. 3 is difference purifying in the embodiment of the present invention 1.
The laver amylose that Fig. 4 is difference purifying in the embodiment of the present invention 2.
The specific IgE level that Fig. 5 is the mouse allergic model sensitization stage.
Fig. 6 is mice serum IgG antibody 1/IgG2a ratio.
The protein level detected result that Fig. 7 is IFN-γ in the mouse spleen lymphocyte culture supernatant.
The protein level detected result that Fig. 8 is IL-4 in the mouse spleen lymphocyte culture supernatant.
The relative expression quantity detected result that Fig. 9 is IFN-γ gene in mouse spleen lymphocyte.
The relative expression quantity detected result that Figure 10 is IL-4 gene in mouse spleen lymphocyte.
Embodiment
The preparation method of polysaccharide in embodiment 1. porphyra haitanensis cakes comprises the following steps:
(1) pre-treatment of laver cake: selecting commercially available porphyra haitanensis cake is raw material;
(2) dry and pulverize: the porphyra haitanensis cake being positioned in 40 ℃ of baking ovens and drying, be ground into Powdered with pulverizer;
(3) hot water extraction: the porphyra haitanensis powder being dissolved in distilled water of weighing, (W: V) be 1: 40,4h are extracted in 100 ℃ of water-baths to solid-to-liquid ratio;
(4) tissue mashing 3min, supernatant is obtained in centrifugal rear filtration;
(5) low pressure is concentrated: adopting Rotary Evaporators (60 ℃, vacuum tightness 0.09MPa) concentrated supernatant is original volume 1/3;
(6) ethanol precipitation: add 4 times of volumes, 95% ethanol precipitation, standing over night, absolute ethanol washing precipitation 3 times, filter and obtain precipitation, vacuum lyophilization through silk;
(7) will precipitate that to redissolve in distilled water to solubility be 20mg/mL, and be splined on the DEAE-cellulose52 chromatography column, and first wash with except impurity such as Deproteinization, pigments with the distillation current, and then adopt 2M NaCl wash-out, flow rate control is at 0.5mL/min.The anthrone sulfuric acid process is measured the elutriant sugar degree, obtains elution curve (Fig. 1), collects A630 > 0.5 elutriant, distill water dialysis 48h, obtain high-purity laver polysaccharide (Fig. 3) thereby dialyzate is carried out to lyophilize.
The preparation method of embodiment 2. fresh Porphyra haitanensis polysaccharide, different with embodiment 1, concrete steps are as follows:
(1) pre-treatment of fresh porphyra haitanensis: selecting fresh porphyra haitanensis is raw material, requires pollution-free, free from extraneous odour.Clean appended silt and the dirt of laver appearance with clear water;
(2) dry and pulverize: the porphyra haitanensis cleaned up being put in to drying in 50 ℃ of baking ovens, being ground into Powdered with pulverizer;
(3) hot water extraction: the porphyra haitanensis powder being dissolved in distilled water of weighing, (W: V) be 1: 30,5h are extracted in 90 ℃ of water-baths to solid-to-liquid ratio;
(4) tissue mashing 5min, supernatant is obtained in centrifugal rear filtration;
(5) low pressure is concentrated: adopting Rotary Evaporators (60 ℃, vacuum tightness 0.09MPa) concentrated supernatant is original volume 1/2;
(6) add 3 times of volumes, 95% ethanol precipitation, standing over night, absolute ethanol washing precipitation 4 times, filter and obtain precipitation, vacuum lyophilization through silk;
(7) to redissolve in distilled water to solubility be 20mg/mL to precipitation, be splined on the DEAE-cellulose52 chromatography column, first with the distillation current, wash to remove the impurity such as Deproteinization, pigment, then adopt 2M NaCl wash-out, flow rate control is at 0.5mL/min, and the anthrone sulfuric acid process is measured the elutriant sugar degree, obtain elution curve (Fig. 2), collect A630 > 0.5 elutriant, distill water dialysis 72h, obtain high-purity laver polysaccharide (Fig. 4) thereby dialyzate is carried out to lyophilize.
(1) animal experiment method.Adopt no-special pathogen (Specific pathogen free, SPF) level BALB/c mouse, with Crustacean main allergen tropomyosin (Tropomyosin, TM) sensitization mouse, set up irritated animal model.Experiment is divided into phosphate buffered saline buffer (Phosphate Buffered Saline, PBS) control group, TM sensitization group, laver amylose group, every group of 6 mouse.At 7,21 days with TM sensitized mice (PBS control group injection PBS); From 0 day, laver amylose group mouse is injected weekly to 3 laver amyloses (PBS control group, TM sensitization group injection PBS), within the 22nd day, get blood and utilize the method for indirect ELISA to detect mice serum antibody IgE, IgG1 and IgG2a level.The result demonstration, laver amylose can obviously reduce IgE content (Fig. 5) and reduction IgG1/IgG2a ratio (Fig. 6) in sensitized mice serum.
(2) cell experiment method.Get TM sensitization group BALB/c mouse spleen, separate lymphocyte.Utilize PBS, TM, TM+ laver amylose to stimulate and cultivate 72h, centrifugal acquisition cell conditioned medium liquid, detect and respectively organize irritated relevant cell factor IL-4 and IFN-γ (Interferon gama, IFN-γ) expressing quantity.The result demonstration, laver amylose can significantly promote the protein expression level (Fig. 7) of Th1 cytokine IFN-γ, significantly suppresses the protein expression level of Th2 cytokine IL-4 (Fig. 8); Get TM sensitization group BALB/c mouse spleen, separate lymphocyte.Utilize PBS, TM, TM+ laver amylose to stimulate and cultivate 72h, centrifugal acquisition cell precipitation, separation and Extraction RNA also obtains cDNA through reverse transcription, utilizes fluorescent quantitative PCR technique to detect the gene expression amount of above two kinds of cytokines.The result demonstration, laver amylose can increase the gene expression amount (Fig. 9) of Th1 cytokine IFN-γ, reduces the gene expression amount (Figure 10) of Th2 cytokine IL-4.
Claims (10)
1. the preparation method of an antianaphylaxis laver amylose is characterized in that its concrete steps are as follows:
1) laver is dried, then be ground into powder;
2) laver powder step 1) obtained is soluble in water, 80～100 ℃ of water-baths, extracts, and tissue mashing, centrifugal, obtain supernatant;
3) by step 2) the water extraction liquid that obtains is concentrated, adds the ethanol precipitation, by multiple soluble in water after the throw out washing, is splined on the anion-exchange chromatography post, measures the sugar degree of elutriant, collects A630 > 0.5 elutriant;
4) elutriant of step 3) being collected is through distill water dialysis, and vacuum lyophilization, obtain the antianaphylaxis laver amylose.
2. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 1), and described laver adopts edible red algae laver.
3. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 1), and the temperature of described oven dry is 40～50 ℃.
4. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 2) in, the proportioning of described laver powder and water is 1: (30～50), wherein the laver powder is calculated in mass, and water is calculated by volume.
5. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 2) in, the time that described water-bath is extracted is 3～5h, the time of tissue mashing is 3～5min.
6. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 3), described concentrated be to adopt Rotary Evaporators by 25%～50% of water extraction liquid simmer down to original volume.
7. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 3), and described to add the condition of ethanol precipitation be 95% ethanol that adds 2～4 times of volumes.
8. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 3), and described washing can adopt absolute ethanol washing precipitation 2～4 times.
9. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 2) and step 3) in, described hydromining distilled water.
10. a kind of preparation method of antianaphylaxis laver amylose as claimed in claim 1, is characterized in that in step 4), and the time of described dialysis is 48～72h.
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|---|---|---|---|---|
| CN109134682A (en) * | 2018-09-14 | 2019-01-04 | 集美大学 | A kind of red algae oligosaccharide and the preparation method and application thereof |
| CN111138556A (en) * | 2020-01-10 | 2020-05-12 | 集美大学 | Antiallergic sulfated polysaccharide and preparation method and application thereof |
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| JP2005126429A (en) * | 2003-09-30 | 2005-05-19 | Japan Science & Technology Agency | Immunopotentiating agent, food and liquid food composition |
| CN102334702A (en) * | 2011-07-12 | 2012-02-01 | 集美大学 | Production method for laver protein and polysaccharide nutrient powder |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005126429A (en) * | 2003-09-30 | 2005-05-19 | Japan Science & Technology Agency | Immunopotentiating agent, food and liquid food composition |
| CN102334702A (en) * | 2011-07-12 | 2012-02-01 | 集美大学 | Production method for laver protein and polysaccharide nutrient powder |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134682A (en) * | 2018-09-14 | 2019-01-04 | 集美大学 | A kind of red algae oligosaccharide and the preparation method and application thereof |
| CN109134682B (en) * | 2018-09-14 | 2021-11-05 | 集美大学 | Red algae oligosaccharide and preparation method and application thereof |
| CN111138556A (en) * | 2020-01-10 | 2020-05-12 | 集美大学 | Antiallergic sulfated polysaccharide and preparation method and application thereof |
| CN111138556B (en) * | 2020-01-10 | 2021-07-20 | 集美大学 | Antiallergic sulfated polysaccharide and preparation method and application thereof |
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| Publication number | Publication date |
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| CN103467616B (en) | 2015-12-23 |
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