CN104814985B - A kind of application of algal polysaccharides - Google Patents
A kind of application of algal polysaccharides Download PDFInfo
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Abstract
The invention belongs to field of biomedicine technology, the application of specifically a kind of algal polysaccharides.Algal polysaccharides are as preparing antiviral or immune preparation.Cell model and interior animal experiment show that algal polysaccharides can significantly improve animal immunizing power, while can promote the generation of cell factor, the parting of T lymphocyte and the proliferation of Mouse spleen cells, so that active cell is immunoreacted.Function algal polysaccharides of the present invention are the natural polysaccharides extracted from the seaweed such as tangleweed kelp, sargassum, centipede algae, Eucheuma, U. pertusa, Enteromorpha, asparagus, yellow tang, tawny daylily algae, are also possible to that algal polysaccharides are degraded obtained low molecular weight seaweed polysaccharide or alga oligosaccharides through different preparation methods.The polyoses extract of single seaweed or the polyoses extract mixture of a variety of seaweed can be used as a kind of novel antiviral and immunopotentiator and be applied to livestock and poultry, in fishes and shrimps shellfish feed, be with a wide range of applications.
Description
Technical field
The invention belongs to field of biomedicine technology, the application of specifically a kind of algal polysaccharides.
Background technique
With the rapid development of aquaculture industry of China, especially intensive aquaculture industry be have developed rapidly in recent years, currently, livestock and poultry disease
Disease prevention and treatment also faces many problems.Livestock and poultry infectious disease can be substantially divided into according to its pathogenic characteristic viral disease, bacteriosis,
Fungal disease, mycoplasmosis etc..And viral disease is to there is no specific medicament and treatment method " difficulty " at present.And it is current
It for the measure that viral infection disease is mainly taken is vaccinated, with treatment methods such as antiviral drugs.Both sides
Method all haves the defects that certain, and vaccine injection effectively, does not have broad spectrum activity just in a certain virus, and uses antiviral
Drug, which is treated, also has certain limitation, they can inhibit common virus, but not to the resistance of some viruses
Enough ideals, and long-time service can generate drug resistance.
Infectious bursa diseases virus vaccine, newcastle disease vaccine can be improved as immunopotentiator in research report, polysaccharide
Effect.Algal polysaccharides are found to have various biological activity, and the activity for having disclosed report at present includes algal polysaccharides
With anti-oxidant, antitumor, antibacterial, antiviral, anticoagulation isoreactivity, but for algal polysaccharides as immunopotentiator, especially
It is that its purposes in terms of preventing and treating avian influenza virus has not been reported.Relative to oil emu inactivated virus vaccine
Several advantages such as speech, algal polysaccharides are abundant with raw material sources, and production cost is low, and preparation process is simple, and it does not have animal body
There is injury, and has improve animal immunizing power, antiviral, anti-oxidant, antibacterial and other effects simultaneously.
Summary of the invention
The purpose of the present invention is to provide a kind of applications of algal polysaccharides.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of application of algal polysaccharides, algal polysaccharides are as preparing antiviral or immune preparation.
The algal polysaccharides are as the virus for preparing anti-livestock and poultry cultivation animal or its immune preparation.
The algal polysaccharides are used as and prepare anti-avian influenza virus, newcastle disease virus, infectious bursa diseases virus, infectiousness
The preparation of bronchitis virus or rotavirus.
The algal polysaccharides are as preparation enhancing T cell or spleen lymphocyte proliferation and active enhancing preparation.
The algal polysaccharides are from kelp, sargassum, thallus laminariae, yellow tang, bladder-wrack, centipede algae, Eucheuma, Dracaena
The Thick many candies extracted in one or more of dish, fragrant plant mentioned in ancient texts, agar, agar, tawny daylily algae, Enteromorpha and U. pertusa tangleweed；
Or, will be from kelp, sargassum, thallus laminariae, yellow tang, bladder-wrack, centipede algae, Eucheuma, asparagus, fragrant plant mentioned in ancient texts, stone
The Thick many candies extracted in one or more of cauliflower, agar, tawny daylily algae, Enteromorpha and U. pertusa tangleweed are by way of degradation
The low-molecular-weight polysaccharide or oligosaccharides of acquisition.
Above-mentioned acquisition algal polysaccharides are made of monosaccharide, predominantly rhamnose, mannose, galactolipin, fucose, xylose, Ah
Uncle sugar, glucose, uronic acid, protein, K, Na, Ca and Mg are drawn, sulfate radical content is between 2%-40%, wherein raw sugar point
Son amount is 400KDa-1000KDa, and average molecular weight is within the scope of 1KDa-400KDa after degrading.
The present invention has the advantage that:
Algal polysaccharides in the present invention derive from large-scale economical alga, and raw material sources are abundant, and production cost is low, and sea
Polysaccharides belong to green product, and the harmful effect being not present to cultivated animals is used for a long time；By cell model and in vivo
Animal experiments show that obtained algal polysaccharides have apparent antivirus action and immunological enhancement, it can be to a variety of
Virus has therapeutic effect, therefore has broad spectrum activity.
It is directly acted on using the algal polysaccharides in the present invention by the cell of virus infection, chicken embryo, can reflect that proof should
Algal polysaccharides have antivirus action.It is used by polysaccharide concentration 1mg/ml or 0.2mg/ml, directly acts on cell or chicken embryo.Sea
Polysaccharides can significantly inhibit the activity of H9N2 influenza virus and infectiousness French capsulitis B87 virus, if hemagglutinative titer reduces, disease
Malicious copy number reduces, and cytokine-expressing increases etc..
Algal polysaccharides of the present invention adjust animal body fluid immunity (see embodiment 2).To be injected intraperitoneally as method, utilization is above-mentioned
Algal polysaccharides are raw material, it may be verified that immunoregulation effect of the polysaccharide to antibody response.By animal (meiofauna) weight ratio
10mg/kg or 50mg/kg is used.After algal polysaccharides and avian influenza virus (AIV) inactivation of viruses mix, by normal immunological dosage,
Immune animal is injected intraperitoneally.It is characterized by: algal polysaccharides effect is more preferable compared with vaccine, as antibody level is promoted significantly, together
When immunoregulation effect it is obvious.
Algal polysaccharides can also enhance the adjustment effect of animal cell immunity power simultaneously (see embodiment 3).Utilize the present invention
In algal polysaccharides and vaccine synergy, i.e., the provable algal polysaccharides there is the work for adjusting cell-mediated immune response
With.It is used by animal (meiofauna) weight ratio 10mg/kg or 50mg/kg.After algal polysaccharides and AIV inactivation of viruses mix, press
Immune animal is injected intraperitoneally in normal immunological dosage.It is characterized by: algal polysaccharides effect is more preferable compared with vaccine, as cell because
Son generates horizontal raising, and the parting of T lymphocyte improves, and promotes spleen cell growth activity etc..
Detailed description of the invention
Fig. 1 be polysaccharide body provided in an embodiment of the present invention outside anti-H9N2 viral hemoagglutination potency figure, wherein asterisk indicate and it is right
There are significant difference (the same below) according between.
Fig. 2 is anti-H9N2 virus H9N2 relative expression's spirogram outside polysaccharide body provided in an embodiment of the present invention.
Fig. 3 is polysaccharide provided in an embodiment of the present invention to viral blocking effect versus cell activity figure.
Fig. 4 is polysaccharide provided in an embodiment of the present invention to viral inhibition versus cell activity figure.
Fig. 5 is polysaccharide provided in an embodiment of the present invention to the direct killing effect cell activity figure of virus.
Fig. 6 is chicken embryo Antiviral breeding survival rate figure provided in an embodiment of the present invention.
Fig. 7 is chicken embryo Antiviral breeding hemagglutinative titer figure provided in an embodiment of the present invention.
Fig. 8 is chicken embryo Antiviral breeding cell factor IL-4 expression figure provided in an embodiment of the present invention.
Fig. 9 is chicken embryo Antiviral breeding cell factor IFN-γ expression figure provided in an embodiment of the present invention.
Figure 10 is that AIV specific antibody contains spirogram in the body of immunized mice twice provided in an embodiment of the present invention.
Figure 11 is that cell factor IFN-γ provided in an embodiment of the present invention contains spirogram.
Figure 12 is that cell factor IL-4 provided in an embodiment of the present invention contains spirogram.
Figure 13 is that T lymphocyte parting CD3+CD4+ provided in an embodiment of the present invention contains spirogram.
Figure 14 is that T lymphocyte parting CD3+CD8+ provided in an embodiment of the present invention contains spirogram.
Figure 15 is various concentration seaweed Thick many candies provided in an embodiment of the present invention to lymphopoietic influence diagram.
Specific embodiment
Embodiment 1
Algal polysaccharides are extracted by taking centipede algae, U. pertusa and sargassum as an example respectively:
Broken centipede algae (Grateloupia filicina) 100g is taken, 5000g distilled water is added, in 100 DEG C of water
It is extracted 2 hours in bath；Filter residue is filtered out with 100 mesh, 200 mesh and 300 mesh thin,tough silk respectively, with reduced pressure after filtrate dialysis desalination
Equipment carries out 1/10 that concentration is original volume, with ethyl alcohol alcohol precipitation 24 hours of the 95% of 4 times of volumes, centrifugation, precipitating freeze-drying to get
Centipede polysaccharides, for use.
Broken U. pertusa (Ulva Pertusa) plus 40 times of distilled water, extract 4 hours in 125 DEG C of water-baths；Point
Filter residue is not filtered out with 100 mesh, 200 mesh and 300 mesh thin,tough silk, it is former for carrying out concentration with reduced pressure equipment after filtrate dialysis desalination
The 1/10 of volume, with ethyl alcohol alcohol precipitation 24 hours of the 95% of 4 times of volumes, centrifugation, precipitating freeze-drying is to get U. pertusa polysaccharide, for use.
3000g distilled water is added in sargassum (Sargassum qingdaoense) 100g, and it is small that 4 are extracted in 91 DEG C of water-baths
When.Filter residue is filtered out with 100 mesh, 200 mesh and 300 mesh thin,tough silk respectively, filtrate dialysis desalination after with reduced pressure equipment carry out it is dense
It is condensed to the 1/10 of original volume, with ethyl alcohol alcohol precipitation 24 hours of the 95% of 4 times of volumes, centrifugation, precipitating freeze-drying is more to get sargassum
Sugar, for use.
Respectively by centipede algae, U. pertusa and sargassum extract algal polysaccharides for further obtain low molecular weight polysaccharide or
Oligosaccharides:
The above-mentioned polysaccharide (raw sugar) for obtaining different algaes respectively is prepared into 0.1%-4% aqueous solution with water respectively, thereto according to
The secondary final concentration of 0-2mol/L and final concentration of 1-10% of hydrogen peroxide of addition hydrochloric acid, it is then auxiliary in the microwave that power is 50-1000W
It helps down, with 50-90 DEG C of degradation 5-60min, solution is neutralized to neutrality with lye after reaction, and dialysis, alcohol precipitation, is collected by centrifugation concentration
Precipitating is freeze-dried the algal polysaccharides up to average molecular weight low molecular weight in each tangleweed within the scope of 1KDa-400KDa
Or oligosaccharides.
The monosaccharide of above-mentioned gained algal polysaccharides forms, predominantly rhamnose, mannose, galactolipin, fucose, xylose, Ah
Uncle sugar, glucose, uronic acid, protein, K, Na, Ca and Mg are drawn, sulfate radical content is between 2%-40%.
Embodiment 2
Antiviral study in vitro
1) anti-H9N2 avian influenza virus
Madin-Darby canine kidney (MDCK) cell is containing 10% tire ox blood in 24 porocyte culture plates
After growing up to single layer in clear DMEM culture medium, culture solution is discarded, inoculation H9N2 virus, is adsorbed one hour after being rinsed twice with PBS
After discard virus, after PBS is rinsed twice plus above-described embodiment containing 100ug/ml or 20ug/ml obtains algal polysaccharides and 1%
Polysaccharide is not added in the DMEM culture medium of fetal calf serum, negative control group.37 DEG C, 5%CO2It is cultivated for 24 hours in environment.Culture solution is taken to measure
Hemagglutinative titer (Hemagglutination test, HA) extracts cell RNA after rinsing twice, fixed by fluorescence after reverse transcription
It measures PCR and detects H9N2 expression quantity.After experiment shows that above-described embodiment, which is added, obtains algal polysaccharides, the hemagglutinative titer of H9N2 has
Certain reduction, and the expression quantity of H9N2 has significant decrease.Wherein 0.2mg/ml U. pertusa polysaccharide and 1mg/ml sargassan
The hemagglutinative titer of processing group has significant decrease, remaining processing group also has certain reduction, sees attached drawing 1.In quantitative fluorescent PCR,
The H9N2 expression quantity of 1mg/ml centipede algae processing group is 0.26 times of negative control group, the centipede algae processing group of 0.2mg/ml
H9N2 expression quantity is 0.20 times of negative control group, illustrates that centipede polysaccharides have obviously inhibiting effect to H9N2 virus；
The H9N2 expression quantity of 1mg/ml U. pertusa processing group is 0.40 times of negative control group, the U. pertusa processing group of 0.2mg/ml
H9N2 expression quantity is 0.23 times of negative control group, illustrates that U. pertusa polysaccharide significantly inhibits H9N2 virus；
The H9N2 expression quantity of 1mg/ml sargassum processing group is 0.64 times of negative control group, the sargassum processing group of 0.2mg/ml
H9N2 expression quantity is 0.49 times of negative control group, illustrates that sargassan has significant inhibiting effect to H9N2 virus, sees
Attached drawing 2.
2) infectivity resistant bursal inflammation B87 virus
Vero cell is subjected to had digestive transfer culture by a conventional method, cell number is adjusted to 1 × 106It is thin to add to 96 holes by a/ml
In born of the same parents' culture plate, the hole 100ul/ (every hole 1 × 105It is a), 5%CO237 DEG C of incubator are cultivated for 24 hours, after cell grows into single layer,
It measures above-described embodiment and obtains algal polysaccharides for blocking effect, inhibiting effect and the direct killing effect of virus.
Blocking effect: above-described embodiment acquisition algal polysaccharides are diluted to 3 dilutions respectively on the basis of safe concentration
It spends (1mg/ml, 0.5mg/ml, 0.2mg/ml), is added in 96 porocyte culture plates for growing up to Vero cell monolayer, the hole 100ul/,
Each dilution repeats 4 holes.37 DEG C of effect 4h, discard medical fluid, and 100TCID is added in every hole50Virus liquid 100ul, 37 DEG C of absorption
1.5h discards virus liquid, is washed 2 times with PBS liquid, and the DMEM culture medium for containing 1% fetal calf serum is added, separately sets cell controls and virus
Control group.37 DEG C, 5%CO2Cell activity is surveyed with mtt assay after culture 48h in incubator.See attached drawing 3.
Inhibiting effect: by 100CID50In poison disease vaccination to 96 porocyte culture plates for the Vero for growing up to single layer, 100ul/
Hole, 37 DEG C of absorption 1.5h, discards virus liquid, is washed 2 times with PBS liquid, and above-described embodiment acquisition algal polysaccharides are diluted to 3 respectively
Dilution (1mg/ml, 0.5mg/ml, 0.2mg/ml), the hole 100ul/, each dilution repeat 6 holes, it is another be arranged cell controls and
Virus control.37 DEG C, 5%CO2Cell activity is measured with mtt assay after culture 48h in incubator.The result shows that above-mentioned reality is added
After applying example acquisition algal polysaccharides, cell activity is improved compared to virus control group, but is still below cell controls group.See attached drawing 4.
Direct killing effect: by above-described embodiment acquisition algal polysaccharides be diluted to respectively 3 dilutions (1mg/ml,
0.5mg/ml, 0.2mg/ml), respectively with isometric 100TCID50Virus liquid mixing, 37 DEG C of effect 2h are added to and grow up to single layer
In 96 porocyte culture plates of vero cell, the hole 100ul/, each dilution repeats 4 holes, separately sets cell controls and virus control,
37 DEG C, 5%CO2After cultivating 48h in incubator, recording method is same as above.As a result polysaccharide is equally shown directly to kill with certain
The effect for virus of going out.See attached drawing 5.
3) interior resisting virus acts on
Polysaccharide can promote chicken embryo to resist virus, based on this, three kinds of extraction above-described embodiments be obtained algal polysaccharides
(U. pertusa polysaccharide, sargassan and centipede polysaccharides) are respectively with tri- kinds of various concentrations of 10g/L, 5g/L, 1g/L and bird flu
H9N2 virus 100EID50Dilution mixed in equal amounts takes above-described embodiment to obtain algal polysaccharides mixed with virus after 37 DEG C of incubation 2h
And liquid, positive and negative control is done respectively with physiological saline and viral dilution, is inoculated in the allantois of 9-10 age in days SPF chicken embryo
Chamber, every inoculation 0.2ml, 37 DEG C of culture 72h observe chicken embryo survival condition；It takes allantoic fluid to survey hemagglutinative titer, extracts RNA, measurement
The expression of cell factor.Test result is as follows: sargassan is in the case where concentration is 5m g/mL, embryo rate highest living,
It is 80%, it is 40% that centipede polysaccharides embryo rate living when concentration is 0.2mg/mL is minimum, and embryo rate of living under the conditions of other is
50% or more, hence it is evident that be better than viral group, see attached drawing 6.Hemagglutinative titer detection display, 0.2mg/mL U. pertusa polysaccharide processing group reduce
It is the most significant, 3 titres are reduced, remaining processing group reduces in 1-2 titre, illustrate that polysaccharide has certain antivirus action,
See attached drawing 7.RNA is extracted, detects the content of IL-4 and IFN-γ in chicken embryo, the results showed that three kinds of polysaccharide can significantly improve cell
The expression of the factor, wherein sargassan improves most obvious in 5mg/mL concentration, sees attached drawing 8,9.It is more in its sargassan
The antiviral effect of sugar is better than U. pertusa polysaccharide and centipede algae.
3 animal immune of embodiment and humoral immunity effect
Polysaccharide can promote the proliferation of B cell, differentiation, so as to cause the generation of antibody.Based on this, by 80 6 weeks
Age kunming mice is randomly divided into 8 groups, every group 10.With final concentration be respectively the centipede algae of 10mg/kg and 50mg/kg, U. pertusa,
It is immunized twice respectively in a manner of intraperitoneal inoculation after sargassan and the mixing of H9N2 avian influenza inactivation virus, while setting two
Group control group, individually injects PBS and inactivation of viruses, and respectively 14 days after being immunized twice, blood sampling separates serum.By with enzyme-linked
Immunoabsorption measures serum AIV specific antibody to evaluate influence of the algal polysaccharides to body's immunity.Pass through the experiment
It can be found that the generation of polysaccharide energy obvious stimulation specific antibody, and it is closely related with dosage.See attached drawing 10.
4 test for celluar immunity of embodiment
1) cell immune response detects:
There are many forms of expression of cellular immunity, and wherein cell factor plays an important role in immune response, adjustable
A variety of reactions.Spleen lymphocyte proliferation and T cell hypotype etc. are also the common counter of cell immune response.
2) cytokines measurement:
Using ELISA method, with the content of IL-4 and IFN-γ in kit (Longtun, China) detection serum.?
It is immune 14 days latter to second of mouse, blood sampling separation serum.By specification operation.The result shows that algal polysaccharides can promote to be immunized
The secretion of relevant cell factor, and effect is different.For IFN-γ, centipede polysaccharides, U. pertusa polysaccharide and sargassan
The polysaccharide effect of 10mg/kg group is better than 50mg/kg group, and processing group is all remarkably higher than vaccine control group and PBS control group, sees
Attached drawing 11.And for IL-4, the effect of 50mg/kg centipede polysaccharides, U. pertusa polysaccharide and sargassan is above 10mg/
Kg, and processing group is above vaccine control group and PBS control group, sees attached drawing 12.
3) T cell parting detects:
T cell hypotype is detected using three-color process.Immune 14 days latter, the blood sampling at second.PE, FITC and PE-Cy5 mark is added
CD3, CD4 and CD8 monoclonal antibody (eBioscience, USA) room temperature of note act on 30 minutes, divide on flow cytometry (BD, LSR)
Analyse cell subsets.The result shows that the differentiation of algal polysaccharides energy obvious stimulation T cell, and it is more bright to CD3+CD4+ inducing effect
Aobvious, effect is slightly above 10mg/kg when centipede polysaccharides, U. pertusa polysaccharide and sargassan concentration are 50mg/kg, sees attached drawing
13,14.
4) spleen lymphocyte proliferation is tested:
Sterile separating mouse spleen lymphocyte is added with the RPMI1640 culture medium suspension cell for containing 10% fetal calf serum
It is respectively 20ug/ml that polysaccharide sample, which causes final concentration, is cultivated after 100ug/ml, 500ug/ml, measures reagent with CCK8 cell number
The relative populations of box survey mouse spleen lymphocyte.The result shows that mice spleen lymph is thin when centipede polysaccharides concentration is 20 μ g/ml
The quantity of born of the same parents is 1.5 times of control group, and when the concentration of centipede polysaccharides is 100 μ g/ml and 500 μ g/ml, mice spleen leaching
There is no significant differences when the quantity of bar cell than polysaccharide concentration is 20 μ g/ml.Illustrate that centipede polysaccharides have extraordinary exempt from
Epidemic disease humidification.When U. pertusa polysaccharide concentration is 20 μ g/ml, the quantity of mouse spleen lymphocyte is 1.4 times of control 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 control group.
Illustrate that U. pertusa polysaccharide has certain immunological enhancement.When sargassan concentration is 20 μ g/ml, mouse spleen lymphocyte
Quantity be 1.6 times of control group, when the concentration of sargassan is 100 μ g/ml, the quantity of mouse spleen lymphocyte is pair
According to 1.9 times of group, when the concentration of sargassan is 500 μ g/ml, the quantity of mouse spleen lymphocyte is the 2.5 of control group
Times.Illustrate that sargassan has extraordinary immunological enhancement, and immunoenhancement result increases with horse hair concentration of algae and increased
Add.See attached drawing 15.
Claims (1)
1. a kind of algal polysaccharides are preparing the application in anti-avian influenza virus or infectious bursa diseases virus preparation；
The algal polysaccharides are the algal polysaccharides extracted from one or more of centipede algae, U. pertusa or sargassum.
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| CN106983761A (en) * | 2015-09-23 | 2017-07-28 | 潘崇良 | Application of the combination of agar oligosaccharides and Qinghai dish oligosaccharides in anti-enterovirus |
| CN105294871B (en) * | 2015-09-25 | 2018-06-22 | 广东医学院 | A kind of preparation method and application with enhancing immune function sea grass polysaccharide |
| CN105533167A (en) * | 2015-12-08 | 2016-05-04 | 福建省农业科学院中心实验室 | Alga oligosaccharide fish feed additive |
| CN105646725B (en) * | 2016-01-22 | 2018-06-05 | 山东畜牧兽医职业学院 | Purposes of the sea grass polysaccharide in fowl poultry immune reinforcing agent is prepared |
| CN106727623B (en) * | 2016-12-21 | 2020-03-17 | 中国科学院海洋研究所 | Application of seaweed oligosaccharide in preparation of anti-avian leukosis virus preparation |
| CN108164614B (en) * | 2018-01-29 | 2020-07-07 | 上海海洋大学 | Preparation method of gelidium amansii polysaccharide with immunoregulation effect, structural part characterization and application thereof |
| CN108477408B (en) * | 2018-04-02 | 2021-04-16 | 中国科学院亚热带农业生态研究所 | Feed for sows at late gestation period for reducing incidence rate of intrauterine growth retardation of fetuses |
| CN111713654A (en) * | 2020-07-27 | 2020-09-29 | 大连工业大学 | Leisure food containing polysaccharide and fishbone calcium and preparation method thereof |
| CN111903934A (en) * | 2020-08-17 | 2020-11-10 | 大连工业大学 | Preparation method and application of maintenance food suitable for people with weak swallowing function |
| CN111904975B (en) * | 2020-09-04 | 2021-05-11 | 佛山蓝强生物科技有限公司 | Algal polysaccharide composition and preparation method and application thereof |
| CN114831311A (en) * | 2022-04-12 | 2022-08-02 | 中国科学院海洋研究所 | Application of red algae starch |
| CN116120482B (en) * | 2023-01-16 | 2024-05-03 | 华南理工大学 | Fucoidan degraded by dielectric barrier discharge plasma, and preparation method and application thereof |
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