CN104814985A - Application of seaweed polysaccharides - Google Patents
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Abstract
The invention belongs to the technical field of biomedicines, and in particular relates to application of seaweed polysaccharides. The seaweed polysaccharides can be taken as preparations for preparing anti-viral or immune medicines. Cell models and in-vivo animal experiments show that the seaweed polysaccharides can be used for significantly enhancing the animal immunity, and can also be used for promoting the generation of cell factors, the typing of T lymphocytes and the proliferation of mouse spleen cells so as to ensure that cellular immune reaction can be activated. The functional seaweed polysaccharides disclosed by the invention are natural polysaccharides which are extracted from seaweeds such as large seaweeds and kelps, gulfweeds, grateloupia sparsa, eucheuma muricatum, ulva pertusa kjellm, enteromorpha, gracilaria, ascophyllum nodosum and scytosiphon lomentaria, and can also be low-molecular-weight seaweed polysaccharides or seaweed oligosaccharides which are prepared by degrading the seaweed polysaccharides by virtue of different preparation methods. According to the application disclosed by the invention, a polysaccharide extract of single seaweed or a polysaccharide extract mixture of a variety of seaweeds can be taken as a novel anti-viral and immune enhancing agent to be applied to feeds of livestock, poultry, fish, shrimps and shellfish, and has wide application prospects.
Description
Technical field
The invention belongs to field of biomedicine technology, specifically a kind of application of Sargassum polysaccharides.
Background technology
Along with the develop rapidly of China's aquaculture, particularly intensive culture industry development is in recent years very fast, and at present, livestock and poultry disease control and prevention also faces a lot of problem.Poultry infectious disease can be divided into viral disease, bacterial disease, fungal disease, mycoplasma etc. substantially according to its pathogenic characteristic.And viral disease there is no specific medicament and Therapeutic Method " difficulty " at present.And the measure mainly taked for viral infection disease is at present vaccinate, uses the Therapeutic Method such as antiviral drugs.All there is certain defect in these two kinds of methods, it is effective that vaccine injection is only aimed at a certain virus, not there is broad spectrum activity, and use antiviral drugs to carry out treating and also have certain limitation, they can suppress common virus, but not ideal enough to the resistance of some virus, and life-time service can produce drug resistance.
Research report, polysaccharide can improve the effect of infectious bursa diseases virus vaccine, newcastle disease vaccine as immunostimulant.Sargassum polysaccharides has been found to have various biological activity, the activity openly reported at present comprises Sargassum polysaccharides and has antioxidation, antitumor, antibacterial, antiviral, anticoagulation isoreactivity, but for Sargassum polysaccharides as immunostimulant, especially its purposes in prevention and therapy bird flu virus have not been reported.For oil emulsion inactivated virus vaccine, some advantages such as Sargassum polysaccharides has abundant raw material source, and production cost is low, and preparation technology is simple, and it does not injure animal body, and have simultaneously and improve animal immunizing power, antiviral, antioxidation, the effect such as antibacterial.
Summary of the invention
The object of the present invention is to provide a kind of application of Sargassum polysaccharides.
For achieving the above object, the technical solution used in the present invention is:
An application for Sargassum polysaccharides, Sargassum polysaccharides is as the preparation preparing antiviral or immunity.
Described Sargassum polysaccharides is as the anti-virus of livestock and poultry cultivation animal of preparation or the preparation of its immunity.
Described Sargassum polysaccharides is as the preparation preparing anti-avian influenza virus, Avian pneumo-encephalitis virus, infectious bursa diseases virus, infectious bronchitis virus or rotavirus.
Described Sargassum polysaccharides strengthens the enhancing preparation of T cell or spleen lymphocyte proliferation and activity as preparation.
Described Sargassum polysaccharides is the crude polysaccharides extracted from one or more tangleweeds Thallus Laminariae (Thallus Eckloniae), Alga Sgrgassi Enerves, Thallus Laminariae, yellow tang, Fucus Vesiculosus, Grateloupia filicina (Wulf.), Eucheuma muricatum (Gmel.) Web.Van Bos., Thallus Gracilariae, Gracilaria tenuistipitata, Eucheuma gelatinosum, agar, Scytosiphon lomentarius (Lyngh.)J. Ag., Entermorpha and U. pertusa;
Or, by the crude polysaccharides extracted from one or more tangleweeds in Thallus Laminariae (Thallus Eckloniae), Alga Sgrgassi Enerves, Thallus Laminariae, yellow tang, Fucus Vesiculosus, Grateloupia filicina (Wulf.), Eucheuma muricatum (Gmel.) Web.Van Bos., Thallus Gracilariae, Gracilaria tenuistipitata, Eucheuma gelatinosum, agar, Scytosiphon lomentarius (Lyngh.)J. Ag., Entermorpha and U. pertusa by the low-molecular-weight polysaccharide that obtains of mode of degraded or oligosaccharide.
Above-mentioned acquisition Sargassum polysaccharides is made up of monosaccharide, be mainly rhamnose, mannose, galactose, fucose, xylose, arabinose, glucose, alduronic acid, protein, K, Na, Ca and Mg, sulfate radical content is between 2%-40%, wherein, raw sugar molecular weight is 400KDa-1000KDa, and after degraded, mean molecule quantity is within the scope of 1KDa-400KDa.
The advantage that the present invention has:
Sargassum polysaccharides in the present invention all derives from large-scale economical alga, abundant raw material source, and production cost is low, and Sargassum polysaccharides belongs to Green Product, and life-time service does not exist the injury effect to cultivated animals; Shown by cell model and interior animal experiment, obtain Sargassum polysaccharides there is obvious antivirus action and immunological enhancement, therapeutic effect can be had to multiple virus, therefore there is broad spectrum activity.
Utilize the Sargassum polysaccharides in the present invention to directly act on by the cell of virus infection, Embryo Gallus domesticus, can reflect and prove that this Sargassum polysaccharides has antivirus action.Use by polysaccharide concentration 1mg/ml or 0.2mg/ml, directly act on cell or Embryo Gallus domesticus.Sargassum polysaccharides significantly can suppress the activity of H9N2 influenza virus and infectiousness French capsulitis B87 virus, and as hemagglutinative titer reduces, viral copy number reduces, cytokine-expressing rising etc.
Sargassum polysaccharides of the present invention regulates animal body fluid immunity (see embodiment 2).Be method with lumbar injection, utilize above-mentioned Sargassum polysaccharides for raw material, the immunoregulation effect that this polysaccharide antagonist reacts can be verified.Use by animal (meiofauna) weight ratio 10mg/kg or 50mg/kg.After Sargassum polysaccharides and bird flu virus (AIV) inactivation of viruses mix, by normal immunological dosage, lumbar injection immune animal.It is characterized in that: compared with vaccine, Sargassum polysaccharides better effects if, as antibody horizontal promotes significantly, immunoregulation effect is obvious simultaneously.
Sargassum polysaccharides also can strengthen the regulating action (see embodiment 3) of animal cell immunity power simultaneously.Utilize the Sargassum polysaccharides in the present invention and vaccine synergy, namely this Sargassum polysaccharides provable has the cell-mediated immunoreactive effect of adjustment.Use by animal (meiofauna) weight ratio 10mg/kg or 50mg/kg.After Sargassum polysaccharides and AIV inactivation of viruses mix, by normal immunological dosage, lumbar injection immune animal.It is characterized in that: compared with vaccine, Sargassum polysaccharides better effects if, improve as cytokine produces level, the lymphocytic typing of T improves, and promotes spleen cell growth activity etc.
Accompanying drawing explanation
Fig. 1 to tire figure for polysaccharide In Vitro Anti H9N2 viral hemoagglutination that the embodiment of the present invention provides, and wherein, asterisk represents and there is significant difference (as follows) between contrasting.
The polysaccharide In Vitro Anti H9N2 virus H9N2 relative expression spirogram that Fig. 2 provides for the embodiment of the present invention.
The polysaccharide that Fig. 3 provides for the embodiment of the present invention is schemed viral blocking effect versus cell activity.
The polysaccharide that Fig. 4 provides for the embodiment of the present invention is schemed viral inhibition versus cell activity.
The polysaccharide that Fig. 5 provides for the embodiment of the present invention is to the direct killing action cytoactive figure of virus.
The Embryo Gallus domesticus Antiviral breeding survival rate figure that Fig. 6 provides for the embodiment of the present invention.
The Embryo Gallus domesticus Antiviral breeding hemagglutinative titer figure that Fig. 7 provides for the embodiment of the present invention.
The Embryo Gallus domesticus Antiviral breeding cytokine IL-4 expression figure that Fig. 8 provides for the embodiment of the present invention.
The Embryo Gallus domesticus Antiviral breeding cytokine IFN-γ expression figure that Fig. 9 provides for the embodiment of the present invention.
In twice immunized mice body that Figure 10 provides for the embodiment of the present invention, AIV specific antibody is containing spirogram.
The cytokine IFN-γ that Figure 11 provides for the embodiment of the present invention contains spirogram.
The cytokine IL-4 that Figure 12 provides for the embodiment of the present invention contains spirogram.
The T lymphocyte typing CD3+CD4+ that Figure 13 provides for the embodiment of the present invention contains spirogram.
The T lymphocyte typing CD3+CD8+ that Figure 14 provides for the embodiment of the present invention contains spirogram.
The variable concentrations Sargassum crude polysaccharides that Figure 15 provides for the embodiment of the present invention is to lymphopoietic effect diagram.
Detailed description of the invention
Embodiment 1
Sargassum polysaccharides is extracted respectively for Grateloupia filicina (Wulf.), U. pertusa and Alga Sgrgassi Enerves:
Get the Grateloupia filicina (Wulf.) after fragmentation (Grateloupia filicina) 100g, add 5000g distilled water, lixiviate 2 hours in 100 DEG C of water-baths; Use 100 orders respectively, filtering residue leaches by 200 orders and 300 mesh sieve thin,tough silk, carries out 1/10 of simmer down to original volume, the ethanol precipitate with ethanol with 95% of 4 times of volumes 24 hours after filtrate dialysis desalination with concentrating under reduced pressure equipment, centrifugal, precipitates lyophilizing, obtains Grateloupia filicina (Wulf.) polysaccharide, stand-by.
U. pertusa (Ulva Pertusa) after fragmentation adds the distilled water of 40 times, lixiviate 4 hours in 125 DEG C of water-baths; Use 100 orders respectively, filtering residue leaches by 200 orders and 300 mesh sieve thin,tough silk, carries out 1/10 of simmer down to original volume, the ethanol precipitate with ethanol with 95% of 4 times of volumes 24 hours after filtrate dialysis desalination with concentrating under reduced pressure equipment, centrifugal, precipitates lyophilizing, obtains U. pertusa polysaccharide, stand-by.
Alga Sgrgassi Enerves (Sargassum qingdaoense) 100g adds 3000g distilled water, lixiviate 4 hours in 91 DEG C of water-baths.Use 100 orders respectively, filtering residue leaches by 200 orders and 300 mesh sieve thin,tough silk, carries out 1/10 of simmer down to original volume, the ethanol precipitate with ethanol with 95% of 4 times of volumes 24 hours after filtrate dialysis desalination with concentrating under reduced pressure equipment, centrifugal, precipitates lyophilizing, obtains sargassan, stand-by.
Extract Sargassum polysaccharides for Grateloupia filicina (Wulf.), U. pertusa and Alga Sgrgassi Enerves respectively and obtain low-molecular-weight polysaccharide or oligosaccharide further:
The above-mentioned polysaccharide (raw sugar) obtaining different algae is respectively prepared 0.1%-4% aqueous solution respectively with water, to add hydrochloric acid final concentration wherein be successively 0-2mol/L and hydrogen peroxide final concentration is 1-10%, then be under the microwave-assisted of 50-1000W at power, with 50-90 DEG C of degraded 5-60min, after reaction, solution alkali liquor is neutralized to neutrality, dialysis, concentrated, precipitate with ethanol, centrifugal collecting precipitation, namely lyophilization obtains mean molecule quantity low-molecular-weight Sargassum polysaccharides or oligosaccharide in each tangleweed within the scope of 1KDa-400KDa.
The monosaccharide composition of above-mentioned gained Sargassum polysaccharides, be mainly rhamnose, mannose, galactose, fucose, xylose, arabinose, glucose, alduronic acid, protein, K, Na, Ca and Mg, sulfate radical content is between 2%-40%.
Embodiment 2
Antiviral study in vitro
1) anti-H9N2 bird flu virus
Madin-Darby canine kidney (MDCK) cell is on 24 porocyte culture plates, grow up to monolayer in the DMEM culture medium containing 10% hyclone after, discard culture fluid, inoculation H9N2 virus after rinsing twice with PBS, adsorb and discard virus after one hour, PBS above-described embodiment added after rinsing twice containing 100ug/ml or 20ug/ml obtains the DMEM culture medium of Sargassum polysaccharides and 1% hyclone, and negative control group does not add polysaccharide.37 DEG C, 5%CO
224h is cultivated in environment.Get culture fluid and measure hemagglutinative titer (Hemagglutination test, HA), after rinsing twice, extract cell RNA, by fluorescence quantitative PCR detection H9N2 expression after reverse transcription.Experiment shows, after adding above-described embodiment acquisition Sargassum polysaccharides, the hemagglutinative titer of H9N2 has had certain reduction, and the expression of H9N2 has remarkable reduction.Wherein the hemagglutinative titer of 0.2mg/ml U. pertusa polysaccharide and 1mg/ml sargassan processed group all has remarkable reduction, and all the other processed group also have certain reduction, sees accompanying drawing 1.In quantitative fluorescent PCR, the H9N2 expression of 1mg/ml Grateloupia filicina (Wulf.) processed group is 0.26 times of negative control group, the H9N2 expression of the Grateloupia filicina (Wulf.) processed group of 0.2mg/ml is 0.20 times of negative control group, illustrates that Grateloupia filicina (Wulf.) polysaccharide has obviously inhibitory action to H9N2 virus; The H9N2 expression of 1mg/ml U. pertusa processed group is 0.40 times of negative control group, and the H9N2 expression of the U. pertusa processed group of 0.2mg/ml is 0.23 times of negative control group, illustrates that U. pertusa polysaccharide has obvious inhibitory action to H9N2 virus; The H9N2 expression of 1mg/ml Alga Sgrgassi Enerves processed group is 0.64 times of negative control group, and the H9N2 expression of the Alga Sgrgassi Enerves processed group of 0.2mg/ml is 0.49 times of negative control group, illustrates that sargassan has significant inhibitory action to H9N2 virus, sees accompanying drawing 2.
2) the scorching B87 virus of infectivity resistant bursal
Vero cell is carried out had digestive transfer culture by method routinely, and cell number adjusts to 1 × 10
6individual/ml, adds in 96 porocyte culture plates, (every hole 1 × 10,100ul/ hole
5individual), 5%CO
2incubator 37 DEG C cultivates 24h, becomes after monolayer until Growth of Cells, measures above-described embodiment and obtains Sargassum polysaccharides for the blocking effect of virus, inhibitory action and direct killing action.
Blocking effect: above-described embodiment is obtained Sargassum polysaccharides on the basis of safe concentration and be diluted to 3 dilution factor (1mg/ml respectively, 0.5mg/ml, 0.2mg/ml), be added to and grow up on 96 porocyte culture plates of Vero cell monolayer, 100ul/ hole, each dilution factor repeats 4 holes.37 DEG C of effect 4h, discard medicinal liquid, every hole adds 100TCID
50virus liquid 100ul, 37 DEG C of absorption 1.5h, discard virus liquid, wash 2 times with PBS liquid, add the DMEM culture medium containing 1% hyclone, separately establish cell controls and virus control group.37 DEG C, 5%CO
2cytoactive is surveyed with mtt assay after cultivating 48h in incubator.See accompanying drawing 3.
Inhibitory action: by 100CID
50virus liquid is seeded on the 96 porocyte culture plates of the Vero growing up to monolayer, 100ul/ hole, 37 DEG C of absorption 1.5h, discard virus liquid, wash 2 times with PBS liquid, above-described embodiment is obtained Sargassum polysaccharides and be diluted to 3 dilution factor (1mg/ml respectively, 0.5mg/ml, 0.2mg/ml), 100ul/ hole, each dilution factor repeats 6 holes, separately arranges cell controls and virus control.37 DEG C, 5%CO
2cytoactive is measured with mtt assay after cultivating 48h in incubator.Result shows, after adding above-described embodiment acquisition Sargassum polysaccharides, cytoactive is compared virus control group and is improved, but still lower than cell controls group.See accompanying drawing 4.
Direct killing action: above-described embodiment is obtained Sargassum polysaccharides and be diluted to 3 dilution factors (1mg/ml, 0.5mg/ml, 0.2mg/ml) respectively, respectively with isopyknic 100TCID
50virus liquid mixes, and 37 DEG C of effect 2h, be added on 96 porocyte culture plates of the vero cell growing up to monolayer, 100ul/ hole, each dilution factor repeats 4 holes, separately establishes cell controls and virus control, 37 DEG C, 5%CO
2after cultivating 48h in incubator, recording method is the same.Result shows the effect that polysaccharide has certain direct kill virus equally.See accompanying drawing 5.
3) interior resisting virus effect
Polysaccharide can promote that Embryo Gallus domesticus resists virus, based on this, three kinds are extracted above-described embodiment and obtain Sargassum polysaccharides (U. pertusa polysaccharide, sargassan and Grateloupia filicina (Wulf.) polysaccharide) respectively with 10g/L, 5g/L, 1g/L tri-kinds of variable concentrations and bird flu H9N2 virus 100EID
50diluent mixed in equal amounts, after hatching 2h at 37 DEG C, get above-described embodiment to obtain Sargassum polysaccharides and virus and mix liquid, the positive and negative control is done respectively with normal saline and viral dilution liquid, be inoculated in the allantoic cavity of 9-10 age in days SPF Embryo Gallus domesticus, often only inoculate 0.2ml, cultivate 72h for 37 DEG C, observe Embryo Gallus domesticus survival condition; Get allantoic fluid and survey hemagglutinative titer, extract RNA, measure the expression of cytokine.Result of the test is as follows: sargassan is when concentration is 5m g/mL, and embryo rate of living is the highest, is 80%, Grateloupia filicina (Wulf.) polysaccharide embryo rate alive under concentration is 0.2mg/mL situation is minimum, is 40%, and embryo rate of living under other condition is more than 50%, significantly better than viral group, see accompanying drawing 6.Hemagglutinative titer detection display, 0.2mg/mL U. pertusa polysaccharide processed group reduces the most remarkable, reduces by 3 titres, and all the other processed group reduce all 1-2 titre, illustrate that polysaccharide has certain antivirus action, see accompanying drawing 7.Extract RNA, detect the content of IL-4 and IFN-γ in Embryo Gallus domesticus, result shows that three kinds of polysaccharide all can significantly improve the expression of cytokine, and wherein sargassan improves the most obvious in 5mg/mL concentration, sees accompanying drawing 8,9.In its sargassan, the antiviral effect of polysaccharide is better than U. pertusa polysaccharide and Grateloupia filicina (Wulf.).
Embodiment 3 animal immune and humoral immunization effect
Polysaccharide can promote propagation, the differentiation of B cell, thus causes the generation of antibody.Based on this, by 80 6 week age kunming mice be divided into 8 groups at random, often organize 10.Twice immunity is carried out respectively in the mode of intraperitoneal inoculation after being respectively the mixing of the Grateloupia filicina (Wulf.) of 10mg/kg and 50mg/kg, U. pertusa, sargassan and H9N2 avian influenza inactivation virus with final concentration, establish two groups of matched groups simultaneously, independent injection PBS and inactivation of viruses, respectively latter 14 days of twice immunity, blood sampling, separation of serum.By evaluating the impact of Sargassum polysaccharides on body's immunity by enzyme-linked immunosorbent assay serum AIV specific antibody.The generation of polysaccharide energy obvious stimulation specific antibody can be found by this experiment, and closely related with dosage.See accompanying drawing 10.
Embodiment 4 test for celluar immunity
1) cell immune response detects:
The form of expression of cellular immunization has multiple, and wherein cytokine plays an important role in immunoreation, can regulate multiple reaction.Spleen lymphocyte proliferation and T cell hypotype etc. are also the common counters of cell immune response.
2) cytokines measurement:
Adopt ELISA method, detect the content of IL-4 and IFN-γ in serum with test kit (Longtun, China).To mice second time immunity latter 14 days, blood sampling separation of serum.By specification operates.Result shows, the secretion of Sargassum polysaccharides energy Promote immunity relevant cell factor, and effect is different.For IFN-γ, the polysaccharide effect of the 10mg/kg group of Grateloupia filicina (Wulf.) polysaccharide, U. pertusa polysaccharide and sargassan is all better than 50mg/kg group, and processed group is all significantly higher than vaccine matched group and PBS matched group, sees accompanying drawing 11.And for the effect of IL-4,50mg/kg Grateloupia filicina (Wulf.) polysaccharide, U. pertusa polysaccharide and sargassan all higher than 10mg/kg, and processed group is all higher than vaccine matched group and PBS matched group, sees accompanying drawing 12.
3) T cell typing detects:
Three-color process is adopted to detect T cell hypotype.Latter 14 days of second time immunity, blood sampling.Add CD3, CD4 and CD8 monoclonal antibody (eBioscience, USA) the room temperature effect 30 minutes of PE, FITC and PE-Cy5 labelling, in the upper analysis of cells hypotype of flow cytometry (BD, LSR).Result shows, the differentiation of Sargassum polysaccharides energy obvious stimulation T cell, and comparatively obvious to CD3+CD4+ inducing effect, and when Grateloupia filicina (Wulf.) polysaccharide, U. pertusa polysaccharide and sargassan concentration are 50mg/kg, effect is a little more than 10mg/kg, sees accompanying drawing 13,14.
4) spleen lymphocyte proliferation test:
Aseptic separating mouse spleen lymphocyte, with containing the RPMI1640 culture medium suspension cell of 10% hyclone, add polysaccharide sample and cause final concentration and be respectively 20ug/ml, 100ug/ml, cultivate after 500ug/ml, measure with CCK8 cell number the relative populations that test kit surveys mouse spleen lymphocyte.Result shows, when Grateloupia filicina (Wulf.) polysaccharide concentration is 20 μ g/ml, the quantity of mouse spleen lymphocyte is 1.5 times of matched group, and when the concentration of Grateloupia filicina (Wulf.) polysaccharide is 100 μ g/ml and 500 μ g/ml, does not have significant difference when the number ratio polysaccharide concentration of mouse spleen lymphocyte is 20 μ g/ml.Illustrate that Grateloupia filicina (Wulf.) polysaccharide has extraordinary immunological enhancement.When U. pertusa polysaccharide concentration is 20 μ g/ml, the quantity of mouse spleen lymphocyte is 1.4 times of matched group, and when the concentration of U. pertusa polysaccharide is 100 μ g/ml and 500 μ g/ml, the quantity of mouse spleen lymphocyte is 1.3 times of matched group.Illustrate that U. pertusa polysaccharide has certain immunological enhancement.When sargassan concentration is 20 μ g/ml, the quantity of mouse spleen lymphocyte is 1.6 times of matched group, when the concentration of sargassan is 100 μ g/ml, the quantity of mouse spleen lymphocyte is 1.9 times of matched group, when the concentration of sargassan is 500 μ g/ml, the quantity of mouse spleen lymphocyte is 2.5 times of matched group.Illustrate that sargassan has extraordinary immunological enhancement, and immunoenhancement result increases with Alga Sgrgassi Enerves concentration and increases.See accompanying drawing 15.
Claims (5)
1. an application for Sargassum polysaccharides, is characterized in that: Sargassum polysaccharides is as the preparation preparing antiviral or immunity.
2. by the application of Sargassum polysaccharides according to claim 1, it is characterized in that: described Sargassum polysaccharides is as the anti-virus of livestock and poultry cultivation animal of preparation or the preparation of its immunity.
3., by the application of Sargassum polysaccharides according to claim 2, it is characterized in that: described Sargassum polysaccharides is as the preparation preparing anti-avian influenza virus, Avian pneumo-encephalitis virus, infectious bursa diseases virus, infectious bronchitis virus or rotavirus.
4. by the application of Sargassum polysaccharides according to claim 2, it is characterized in that: described Sargassum polysaccharides strengthens the enhancing preparation of T cell or spleen lymphocyte proliferation and activity as preparation.
5., by the application of Sargassum polysaccharides according to claim 1, it is characterized in that: described Sargassum polysaccharides is the crude polysaccharides extracted from one or more tangleweeds Thallus Laminariae (Thallus Eckloniae), Alga Sgrgassi Enerves, Thallus Laminariae, yellow tang, Fucus Vesiculosus, Grateloupia filicina (Wulf.), Eucheuma muricatum (Gmel.) Web.Van Bos., Thallus Gracilariae, Gracilaria tenuistipitata, Eucheuma gelatinosum, agar, Scytosiphon lomentarius (Lyngh.)J. Ag., Entermorpha and U. pertusa;
Or, by the crude polysaccharides extracted from one or more tangleweeds in Thallus Laminariae (Thallus Eckloniae), Alga Sgrgassi Enerves, Thallus Laminariae, yellow tang, Fucus Vesiculosus, Grateloupia filicina (Wulf.), Eucheuma muricatum (Gmel.) Web.Van Bos., Thallus Gracilariae, Gracilaria tenuistipitata, Eucheuma gelatinosum, agar, Scytosiphon lomentarius (Lyngh.)J. Ag., Entermorpha and U. pertusa by the low-molecular-weight polysaccharide that obtains of mode of degraded or oligosaccharide.
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