CN104814985A - Application of seaweed polysaccharides - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to an application of seaweed polysaccharide. Seaweed polysaccharides are used as preparations for antiviral or immune preparations. Cell models and in vivo animal experiments have shown that seaweed polysaccharides can significantly improve animal immunity, and at the same time promote the production of cytokines, the typing of T lymphocytes and the proliferation of mouse spleen cells, thereby activating cellular immune responses. The functional seaweed polysaccharides involved in the present invention are natural polysaccharides extracted from seaweeds such as large seaweed kelp, sargassum, centipede algae, Eucheuma, Ulva, Enteromorpha, asparagus, Ascophyllum nodosum, and Hemerophyllum. It is a low molecular weight seaweed polysaccharide or seaweed oligosaccharide obtained by degrading seaweed polysaccharide through different preparation methods. The polysaccharide extract of a single type of seaweed or the mixture of polysaccharide extracts of various seaweeds can be used as a new type of anti-virus and immune enhancer in feed for livestock, poultry, fish, shrimp and shellfish, and has broad application prospects.

Description

A kind of application of seaweed polysaccharide

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to an application of seaweed polysaccharide.

Background technique

With the rapid development of my country's aquaculture industry, especially in recent years, the intensive aquaculture industry has developed rapidly. At present, the prevention and control of livestock and poultry diseases is also facing many problems. Infectious diseases of livestock and poultry can be roughly divided into viral diseases, bacterial diseases, fungal diseases, mycoplasma diseases, etc. according to their pathogenic characteristics. Viral diseases are "difficult problems" that do not have specific drugs and methods of treatment at present. At present, the main measures taken for viral infectious diseases are injections, antiviral drugs and other therapeutic methods. These two methods have certain defects. Vaccination is only effective for a certain virus and does not have a broad spectrum. The use of antiviral drugs for treatment also has certain limitations. They can inhibit common viruses, but they are not effective for The resistance of some viruses is not ideal, and long-term use will produce drug resistance.

Studies have reported that polysaccharides as immune enhancers can improve the efficacy of infectious bursal disease virus vaccine and Newcastle disease vaccine. Seaweed polysaccharides have been found to have a variety of biological activities. The activities that have been publicly reported include seaweed polysaccharides having antioxidant, antitumor, antibacterial, antiviral, and anticoagulant activities. However, for seaweed polysaccharides as immune enhancers, especially Its use in the prevention and treatment of avian influenza virus has not been reported yet. Compared with oil-emulsion all-virus inactivated vaccines, seaweed polysaccharide has several advantages such as rich source of raw materials, low production cost, simple preparation process, etc., and it has no harm to animals, and at the same time has the functions of improving animal immunity, anti-virus, anti- Oxidation, antibacterial and other effects.

Contents of the invention

The object of the present invention is to provide a kind of application of seaweed polysaccharide.

To achieve the above object, the technical solution adopted in the present invention is:

The invention relates to an application of seaweed polysaccharide as preparation of antiviral or immune preparation.

The seaweed polysaccharide is used as preparation for anti-virus or immunity of livestock and poultry animals.

The seaweed polysaccharide is used as preparation for resisting avian influenza virus, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus or rotavirus.

The seaweed polysaccharide is used as an enhancing preparation for enhancing the proliferation and activity of T cells or spleen lymphocytes.

The seaweed polysaccharide is obtained from kelp, sargassum, wakame, ascophyllum nodosum, fucus, centipede algae, Eucheuma, asparagus, gracilaria, agarose, agar, Hemerocallis, enteromorpha and Ulva Crude polysaccharides extracted from one or several large seaweeds;

Or, one of kelp, sargassum, wakame, ascophyllum, fucus, centipede, Eucheuma, asparagus, gracilaria, agaric, agar, hemerocallis, enteromorpha and ulva Low molecular weight polysaccharides or oligosaccharides obtained by degrading crude polysaccharides extracted from one or several large seaweeds.

The seaweed polysaccharides obtained above are composed of monosaccharides, mainly rhamnose, mannose, galactose, fucose, xylose, arabinose, glucose, uronic acid, protein, K, Na, Ca and Mg, sulfate content Between 2% and 40%, wherein the molecular weight of the crude sugar is 400KDa-1000KDa, and the average molecular weight after degradation is within the range of 1KDa-400KDa.

The advantages that the present invention has:

The seaweed polysaccharides in the present invention are all derived from large-scale economic seaweeds, which have rich sources of raw materials and low production cost, and seaweed polysaccharides are green and environmentally friendly products, and there is no harmful effect on farmed animals in long-term use; cell models and in vivo animal experiments show that the The obtained seaweed polysaccharide has obvious antiviral effect and immune enhancement effect, and can have therapeutic effect on various viruses, so it has broad spectrum.

Utilizing the seaweed polysaccharide in the present invention directly acts on cells and chicken embryos infected by the virus, it can be reflected and proved that the seaweed polysaccharide has an antiviral effect. Use according to the polysaccharide concentration of 1mg/ml or 0.2mg/ml, directly acting on cells or chicken embryos. Seaweed polysaccharides can significantly inhibit the activity of H9N2 influenza virus and infectious bursitis B87 virus, such as decreased hemagglutination titer, decreased virus copy number, increased expression of cytokines, etc.

The seaweed polysaccharide of the present invention regulates the humoral immunity of animals (see Example 2). Using intraperitoneal injection as a method, using the above-mentioned seaweed polysaccharide as a raw material, the immunomodulatory effect of the polysaccharide on antibody response can be verified. Use according to animal (small animal) weight ratio 10mg/kg or 50mg/kg. After the seaweed polysaccharide is mixed with the inactivated virus of avian influenza virus (AIV), the animal is injected intraperitoneally according to the normal immunization dose. It is characterized in that: compared with the vaccine, the effect of seaweed polysaccharide is better, such as a significant increase in the antibody level, and at the same time, the immune regulation effect is obvious.

At the same time, seaweed polysaccharides can also enhance the regulation of animal cell immunity (see Example 3). Utilizing the joint effect of the seaweed polysaccharide in the present invention and the vaccine, it can be proved that the seaweed polysaccharide has the function of regulating the immune response mediated by cells. Use according to the animal (small animal) body weight ratio of 10mg/kg or 50mg/kg. After the seaweed polysaccharide is mixed with the inactivated AIV virus, the animal is injected intraperitoneally according to the normal immunization dose. It is characterized in that: compared with vaccines, seaweed polysaccharides have better effects, such as increased production of cytokines, improved typing of T lymphocytes, and promotion of growth activity of spleen cells.

Description of drawings

Fig. 1 is the in vitro anti-H9N2 virus hemagglutination potency chart of the polysaccharide provided by the embodiment of the present invention, wherein the asterisk indicates that there is a significant difference from the control (the same applies hereinafter).

Fig. 2 is a graph showing the relative expression level of the polysaccharide against H9N2 virus H9N2 in vitro provided by the embodiment of the present invention.

Fig. 3 is a graph of the blocking effect of polysaccharides on viruses relative to cell activity provided by the embodiment of the present invention.

Fig. 4 is a diagram of the relative cell activity of polysaccharides against viruses provided by the examples of the present invention.

Fig. 5 is a diagram of the cell activity of polysaccharides directly killing viruses provided by the embodiment of the present invention.

Fig. 6 is a graph of the survival rate of chicken embryo anti-virus experiment provided by the embodiment of the present invention.

Fig. 7 is a hemagglutination titer diagram of chicken embryo anti-virus experiment provided by the embodiment of the present invention.

Fig. 8 is a graph showing the expression level of cytokine IL-4 in chicken embryo anti-virus experiment provided by the embodiment of the present invention.

Fig. 9 is a graph showing the expression level of cytokine IFN-γ in the chicken embryo anti-virus experiment provided by the embodiment of the present invention.

Fig. 10 is a diagram of the content of AIV-specific antibodies in mice after two immunizations provided by the embodiment of the present invention.

Fig. 11 is a content map of the cytokine IFN-γ provided by the embodiment of the present invention.

Fig. 12 is a diagram of the content of cytokine IL-4 provided by the embodiment of the present invention.

Fig. 13 is a CD3+CD4+ content map of T lymphocyte typing provided by the embodiment of the present invention.

Fig. 14 is a CD3+CD8+ content map of T lymphocyte typing provided by the embodiment of the present invention.

Fig. 15 is a graph showing the influence of different concentrations of seaweed crude polysaccharides on lymphocyte proliferation provided by the embodiment of the present invention.

Detailed ways

Example 1

Taking centipede algae, Ulva pore and Sargassum as examples to extract seaweed polysaccharides:

Take 100 g of crushed centipede algae (Grateloupia filicina), add 5000 g of distilled water, and extract in a water bath at 100° C. for 2 hours; use 100 mesh, 200 mesh and 300 mesh sieves to filter out the filter residue, and then dialyze the filtrate to desalinate it with reducing Concentrate to 1/10 of the original volume by pressing the concentrating equipment, precipitate with 4 times the volume of 95% ethanol for 24 hours, centrifuge, freeze-dry the precipitate, and obtain the centipede algal polysaccharide, which is ready for use.

Add 40 times of distilled water to the crushed Ulva Pertusa, and extract in a water bath at 125°C for 4 hours; use 100-mesh, 200-mesh and 300-mesh sieves to filter out the filter residue, and then dialyze the filtrate to desalinate it with reducing Concentrate to 1/10 of the original volume by pressing the concentrating equipment, precipitate with 4 times the volume of 95% ethanol for 24 hours, centrifuge, precipitate and freeze-dry to obtain Ulva pore polysaccharide, which is ready for use.

Add 100 g of Sargassum qingdaoense to 3000 g of distilled water, and extract in a water bath at 91° C. for 4 hours. Use 100 mesh, 200 mesh and 300 mesh sieves to filter out the filter residue, and then use a vacuum concentration equipment to concentrate the filtrate to 1/10 of the original volume after dialysis and desalination, and use 4 times the volume of 95% ethanol to precipitate for 24 hours , centrifuged, and the precipitate was freeze-dried to obtain the polysaccharide from Sargassum algae for use.

Taking seaweed polysaccharides extracted from Centipede algae, Ulva pore and Sargassum as examples to further obtain low molecular weight polysaccharides or oligosaccharides:

The polysaccharides (rough sugar) obtained from different algae mentioned above were respectively prepared with 0.1%-4% aqueous solution, and the final concentration of hydrochloric acid was 0-2mol/L and the final concentration of hydrogen peroxide was 1-10%, and then the power was 50- Under the assistance of 1000W microwave, degrade at 50-90°C for 5-60min. After the reaction, the solution is neutralized to neutral with lye, dialyzed, concentrated, alcohol precipitated, centrifuged to collect the precipitate, and freeze-dried to obtain an average molecular weight in the range of 1KDa-400KDa Low molecular weight algal polysaccharides or oligosaccharides in various macroalgae.

The monosaccharide composition of the seaweed polysaccharide obtained above is mainly rhamnose, mannose, galactose, fucose, xylose, arabinose, glucose, uronic acid, protein, K, Na, Ca and Mg, and the sulfate radical content Between 2%-40%.

Example 2

In vitro antiviral experiment

1) Anti-H9N2 avian influenza virus

Madin-Darby canine kidney (MDCK) cells were grown into a monolayer on a 24-well cell culture plate in DMEM medium containing 10% fetal bovine serum, the culture medium was discarded, washed twice with PBS, and then inoculated with H9N2 virus. After one hour of adsorption, the virus was discarded, washed twice with PBS, and DMEM medium containing 100 ug/ml or 20 ug/ml of seaweed polysaccharide and 1% fetal bovine serum obtained from the above-mentioned embodiments was added, and the negative control group did not add polysaccharide. Incubate for 24 hours at 37°C in a 5% CO ₂ environment. The culture medium was taken to measure the hemagglutination titer (Hemagglutination test, HA), the cellular RNA was extracted after washing twice, and the expression level of H9N2 was detected by fluorescent quantitative PCR after reverse transcription. Experiments have shown that after adding the seaweed polysaccharide obtained in the above examples, the hemagglutination titer of H9N2 has been reduced to a certain extent, and the expression level of H9N2 has been significantly reduced. Among them, the hemagglutination titer of 0.2mg/ml Ulva polysaccharide and 1mg/ml Sargassum polysaccharide treatment group were significantly reduced, and the remaining treatment groups also had a certain reduction, see Figure 1. In fluorescent quantitative PCR, the H9N2 expression level of the 1mg/ml centipede algae treatment group was 0.26 times that of the negative control group, and the H9N2 expression level of the 0.2mg/ml centipede algae treatment group was 0.20 times that of the negative control group, indicating that the centipede algae polysaccharide It has a very obvious inhibitory effect on the H9N2 virus; the H9N2 expression level of the 1mg/ml Ulva pore treatment group was 0.40 times that of the negative control group, and the H9N2 expression level of the 0.2mg/ml Ulva pore treatment group was 0.23 times that of the negative control group. times, indicating that Ulva polysaccharides have a significant inhibitory effect on H9N2 virus; the expression of H9N2 in the 1mg/ml Sargassum treatment group was 0.64 times that of the negative control group, and the H9N2 expression in the 0.2mg/ml Sargassum treatment group was negative 0.49 times that of the control group, it shows that Sargassum polysaccharide has significant inhibitory effect on H9N2 virus, see accompanying drawing 2.

2) Anti-infectious bursal disease B87 virus

The Vero cells were digested and passaged according to the conventional method, the cell number was adjusted to 1×10 ⁶ cells/ml, added to a 96-well cell culture plate, 100ul/well (1×10 ⁵ cells per well), cultured in 5% CO ₂ Incubate at 37° C. for 24 hours, and after the cells grow into a single layer, measure the blocking effect, inhibitory effect and direct killing effect of the seaweed polysaccharide obtained in the above embodiment on the virus.

Blocking effect: On the basis of the safe concentration, the seaweed polysaccharide obtained in the above-mentioned embodiment was diluted into 3 dilutions (1mg/ml, 0.5mg/ml, 0.2mg/ml) respectively, and added to 96% Vero cells grown into a monolayer Well cell culture plate, 100ul/well, repeat 4 wells for each dilution. Treat at 37°C for 4 hours, discard the drug solution, add 100ul of 100TCID ₅₀ virus solution to each well, absorb at 37°C for 1.5h, discard the virus solution, wash twice with PBS solution, add DMEM medium containing 1% fetal bovine serum, and add A cell control and a virus control group were set up. Cell viability was measured by MTT method after culturing in 37°C, 5% CO ₂ incubator for 48 hours. See attached picture 3.

Inhibitory effect: Inoculate 100CID ₅₀ virus liquid onto a Vero 96-well cell culture plate grown into a monolayer, 100ul/well, absorb at 37°C for 1.5h, discard the virus liquid, wash twice with PBS, and apply the above-mentioned examples The obtained seaweed polysaccharides were diluted to 3 dilutions (1mg/ml, 0.5mg/ml, 0.2mg/ml), 100ul/well, each dilution was repeated for 6 wells, and a cell control and a virus control were set up. Cell viability was determined by MTT method after culturing in 37°C, 5% CO ₂ incubator for 48 hours. The results showed that after adding the seaweed polysaccharide obtained in the above embodiment, the cell activity was improved compared with the virus control group, but still lower than the cell control group. See attached drawing 4.

Direct killing effect: Dilute the seaweed polysaccharides obtained in the above examples into three dilutions (1mg/ml, 0.5mg/ml, 0.2mg/ml), mix them with an equal volume of 100TCID ₅₀ virus liquid, and act at 37°C for 2h , add to the 96-well cell culture plate of Vero cells growing into a single layer, 100ul/well, repeat 4 wells for each dilution, and set up cell control and virus control, and culture in 37°C, 5% CO ₂ incubator for 48h After that, the recording method is the same as above. The results also show that the polysaccharide has a certain effect of directly killing viruses. See attached drawing 5.

3) Antiviral effect in vivo

Polysaccharides can promote chicken embryos to resist viruses. Based on this, three kinds of seaweed polysaccharides (Ulva polysaccharides, Sargassum polysaccharides and centipede polysaccharides) obtained from the above-mentioned embodiments were extracted at 10g/L, 5g/L, and 1g/L respectively. Three different concentrations were mixed with the avian influenza H9N2 virus 100EID ₅₀ dilution in equal amounts, and after incubation at 37°C for 2 hours, the mixture of seaweed polysaccharide and virus was obtained according to the above example, and normal saline and virus dilution were used as positive and negative controls respectively. Inoculate in the allantoic cavity of 9-10 day old SPF chicken embryos, inoculate 0.2ml each, culture at 37°C for 72 hours, observe the survival of the chicken embryos; take the allantoic fluid to measure the hemagglutination titer, extract RNA, and measure the expression of cytokines level. The test results are as follows: when the concentration of sargassum polysaccharide is 5mg/mL, the live embryo rate is the highest, which is 80%, and when the concentration of centipede algae polysaccharide is 0.2mg/mL, the live embryo rate is the lowest, which is 40%. Other conditions The live embryo rate was more than 50%, which was obviously better than that of the virus group, as shown in Figure 6. The detection of hemagglutination titer showed that the 0.2mg/mL Ulva polysaccharide treatment group decreased most significantly, with a decrease of 3 titers, and the reductions of the other treatment groups were all at 1-2 titers, indicating that the polysaccharide has a certain antiviral effect, see Figure 7. RNA was extracted to detect the contents of IL-4 and IFN-γ in chicken embryos. The results showed that the three polysaccharides could significantly increase the expression of cytokines, and the Sargassum polysaccharides increased most obviously at a concentration of 5 mg/mL. See accompanying drawing 8. 9. The antiviral effect of the polysaccharides in the Sargassum polysaccharides is better than that of Ulva polysaccharides and centipede algae.

Embodiment 3 animal immunity and humoral immunity

Polysaccharides can promote the proliferation and differentiation of B cells, leading to the production of antibodies. On this basis, 80 6-week-old Kunming mice were randomly divided into 8 groups, 10 in each group. The final concentrations of 10mg/kg and 50mg/kg of centipede algae, Ulva pore, Sargassum polysaccharide and H9N2 avian influenza inactivated virus were mixed and immunized twice by intraperitoneal inoculation, and two groups of control groups were set up at the same time. PBS and inactivated virus were injected separately, blood was collected 14 days after the two immunizations, and serum was separated. The effect of seaweed polysaccharides on the immune function of the body was evaluated by measuring the serum AIV specific antibody by enzyme-linked immunosorbent assay. Through this experiment, it can be found that the polysaccharide can significantly stimulate the production of specific antibodies, and it is closely related to the dosage. See attached drawing 10.

Example 4 Cellular Immunity Test

1) Detection of cellular immune response:

There are many manifestations of cellular immunity, among which cytokines play an important role in the immune response and can regulate a variety of responses. Splenic lymphocyte proliferation and T cell subtypes are also commonly used indicators of cellular immune responses.

2) Cytokine detection:

ELISA method was used to detect the contents of IL-4 and IFN-γ in serum with a kit (Longtun, China). Fourteen days after the second immunization of mice, blood was collected to separate serum. Follow the instructions. The results showed that seaweed polysaccharides could promote the secretion of immune-related cytokines with different effects. For IFN-γ, the polysaccharide effects of the 10mg/kg group of centipede polysaccharides, Ulva polysaccharides and Sargassum polysaccharides were better than those of the 50mg/kg group, and the treatment group was significantly higher than the vaccine control group and the PBS control group, see attached Figure 11. For IL-4, the effects of 50mg/kg centipede polysaccharide, Ulva polysaccharide and Sargassum polysaccharide were all higher than 10mg/kg, and the treatment group was higher than the vaccine control group and PBS control group, see Figure 12.

3) T cell typing detection:

T cell subtypes were detected by the three-color method. Fourteen days after the second immunization, blood was collected. PE, FITC and PE-Cy5 labeled CD3, CD4 and CD8 monoclonal antibodies (eBioscience, USA) were added to act at room temperature for 30 minutes, and cell subtypes were analyzed on a flow cytometer (BD, LSR). The results show that seaweed polysaccharide can significantly stimulate the differentiation of T cells, and the induction effect on CD3+CD4+ is more obvious. When the concentration of polysaccharides from centipede algae polysaccharides, Ulva poreus polysaccharides and sargassum polysaccharides is 50 mg/kg, the effect is slightly higher than 10 mg/kg, see Figures 13, 14.

4) Spleen lymphocyte proliferation test:

Aseptically isolate mouse spleen lymphocytes, suspend the cells in RPMI1640 medium containing 10% fetal bovine serum, add polysaccharide samples to a final concentration of 20ug/ml, 100ug/ml, and 500ug/ml, and culture them, and measure them by the number of CCK8 cells The kit measures the relative number of mouse spleen lymphocytes. The results showed that when the concentration of centipede algal polysaccharide was 20 μg/ml, the number of mouse spleen lymphocytes was 1.5 times that of the control group, and when the concentration of centipede algal polysaccharide was 100 μg/ml and 500 μg/ml, the number of mouse spleen lymphocytes There was no significant difference in the number ratio when the polysaccharide concentration was 20 μg/ml. It shows that the centipede algae polysaccharide has a very good immune enhancing effect. When the concentration of Ulva polysaccharides was 20 μg/ml, the number of mouse spleen lymphocytes was 1.4 times that of the control group, and when the concentration of Ulva polysaccharides was 100 μg/ml and 500 μg/ml, the number of mouse spleen lymphocytes 1.3 times that of the control group. It shows that the Ulva polysaccharide has a certain immune enhancing effect. When the concentration of Sargassum polysaccharides was 20 μg/ml, the number of mouse spleen lymphocytes was 1.6 times that of the control group, and when the concentration of Sargassum polysaccharides was 100 μg/ml, the number of mouse spleen lymphocytes was 1.9 times that of the control group. When the concentration of Sargassum polysaccharide was 500μg/ml, the number of mouse spleen lymphocytes was 2.5 times that of the control group. It shows that Sargassum polysaccharide has a very good immune enhancement effect, and the immune enhancement effect increases with the increase of Sargassum concentration. See attached 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.