CN115746155A - Preparation method and application of sargassum polysaccharide extract SP1 - Google Patents
Preparation method and application of sargassum polysaccharide extract SP1 Download PDFInfo
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Abstract
The invention discloses a preparation method of a sargassum polysaccharide extract SP1, which comprises the following steps: (1) Weighing dry Sargassum powder, removing pigment with ethanol, oven drying, adding water, adding papain, transferring to boiling water bath after water bath to inactivate protease, water bath, centrifuging to obtain supernatant, repeatedly extracting precipitate once, mixing the supernatants, and evaporating; (2) Concentrating, precipitating with ethanol, and freeze drying to obtain crude Sargassum polysaccharide extract; (3) Eluting the crude sargassum polysaccharide extract by an elution column to obtain a sargassum polysaccharide extract SP1. The sargassum polysaccharide extract SP1 obtained by the method can achieve the effect of resisting PRRSV infection by inhibiting the adsorption and replication of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in MARC-145 cells and the release of virus particles, and the sargassum polysaccharide extract SP1 obtained by the method can be applied to the preparation of medicines for resisting PRRSV infection.
Description
Technical Field
The invention belongs to the technical field of Chinese herbal medicine sargassum development, and particularly relates to a preparation method and application of sargassum polysaccharide extract SP1.
Background
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the arterivirus family of the order platyphyllales, is an enveloped single-stranded positive-strand RNA virus that infects pigs of various ages with clinical symptoms mainly manifested as anorexia, fever, respiratory and reproductive disturbances, which can lead to lymph node damage and to systemic sepsis. Infected pigs or virus-carrying pigs are an important source of infection of PRRSV, and the disease is widely epidemic due to continuous recombination of wild strains and vaccine strains in clinic, so that the vaccine immune effect is reduced. Meanwhile, the PRRSV can survive in lymph nodes, spleen, lung and other tissues of infected pigs for a long time and continuously expel toxin outwards. Especially, the nursery pigs which are purified from PRRSV negative breeding pig farms are transferred to young pig farms for fattening, and the phenomenon of returning to the positive often occurs, thereby causing the prevalence and spread of diseases. At present, no effective medicine exists for preventing and controlling the disease.
Perennial Sargassum (Sargassum) belongs to Phaeophyta (Phaeophyta), fucales (Fucales) and Sargassaceae (sargasaceae), and is widely distributed in the east and south China sea areas. Sargassum is nutritious, and is rich in polysaccharide, polyphenol, terpenoids and various nutritional components. The sargassum polysaccharide is a polysaccharide which is extracted from sargassum, is easy to dissolve in water and has high viscosity, is one of main chemical components of sargassum, mainly comprises three kinds of polysaccharides such as brown algae, brown algae starch, brown algae polysaccharide sulfate and the like, and has biological activities of oxidation resistance, tumor resistance, blood sugar reduction, inflammation resistance, virus resistance, immune regulation and the like.
The gulfweed contains more pigments and proteins, and the proteins can be efficiently enzymolyzed into amino acids by an enzyme method, so that the proteins are effectively removed. When polysaccharides are prepared, there may be co-present pigments in the extract that may affect its isolation, purification, structural characterization and biological activity. Thus, decolorization is an essential step in the process of preparing plant polysaccharides. By adopting the principle of similar intermiscibility and adopting the method of adding ethanol for evaporation, condensation and reflux to remove pigments, the obtained polysaccharide is white. Most researches show that the sargassum polysaccharide has biological activities of enhancing immunity, resisting oxidation, resisting viruses and the like, the research on the sargassum polysaccharide at present mostly focuses on sulfated polysaccharide or extracted sargassum crude polysaccharide, but the research on the preparation and purification process of the active components of the sargassum polysaccharide is less, in particular to the preparation process of the active components of the sargassum polysaccharide for resisting porcine reproductive and respiratory syndrome viruses.
Disclosure of Invention
Aiming at the technical problems, the invention provides a preparation method and application of sargassum polysaccharide extract SP1, and the prepared sargassum polysaccharide extract SP1 has the characteristics of pure nature, easiness in preparation, low cost, capability of effectively resisting infection and replication of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and the like.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a preparation method of sargassum polysaccharide extract SP1 comprises the following operation steps:
(1) Weighing dry gulfweed powder, removing pigment by using ethanol, drying, and then mixing the powder according to the weight ratio of 1:10 to 1: adding distilled water (namely adding 10ml of distilled water into 1g of gulfweed dry powder) according to the mass ratio of 30, adding papain of which the mass is 0.10-0.5% of that of the gulfweed dry powder, transferring the mixture after water bath to inactivate the protease in boiling water bath, carrying out water bath for 1-4 h at 50-90 ℃, centrifuging to obtain supernatant, repeatedly extracting the precipitate once, combining the supernatants obtained by two times, and evaporating;
(2) Concentrating the evaporated substances obtained in the step (1), precipitating with ethanol, removing protein, dissolving the precipitate with distilled water, and freeze drying to obtain crude Sargassum polysaccharide extract;
(3) Eluting the crude Sargassum polysaccharide extract with an elution column to obtain Sargassum polysaccharide extract distilled water part (SP 1), i.e. Sargassum polysaccharide extract SP1; wherein the elution component is distilled water.
Preferably, the pigment removal by using ethanol in the step (1) is to add ethanol with the volume concentration of 95% and reflux the mixture at 80 ℃ until the ethanol does not change color, and then remove the ethanol by vacuum filtration.
Preferably, the drying in the step (1) is oven drying at 55 ℃.
Preferably, in step (1), after being placed in a water bath at 52 ℃ for 1h, the protease is inactivated by transferring the protease to a boiling water bath at 100 ℃ for 0.5 h.
Preferably, the concentration and alcohol precipitation in the step (2) is to add trichloroacetic acid with the mass concentration of 25% to sequentially stand, centrifuge and concentrate, add ethanol with the volume concentration of 95% to the obtained concentrated solution to precipitate overnight, centrifuge, discard the supernatant, and take the precipitate.
Preferably, the elution column described in step (3) is DEAE-Sepharose Fast Flow or DEAE52.
The sargassum polysaccharide extract SP1 prepared by the method is applied to preparing a medicament for resisting Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
A preparation for resisting Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) contains Sargassum polysaccharide extract SP1 as effective component.
The sargassum polysaccharide extract SP1 prepared by the invention achieves the effect of resisting PPRSV infection by inhibiting the adsorption, replication and release of PRRSV in MARC-145 cells.
Compared with the prior art, the invention has the following beneficial effects:
the sargassum polysaccharide effective component SP1 extract obtained by the method has high yield and high total content, and does not contain starch, amino acid or protein; the sargassum polysaccharide extract SP1 prepared by the invention can achieve the effect of resisting PRRSV infection by inhibiting the adsorption and replication of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in MARC-145 cells and the release of virus particles, and the sargassum polysaccharide extract SP1 prepared by the invention can be applied to the preparation of medicaments for resisting PRRSV infection.
Detailed Description
The following detailed description is to be read in connection with specific embodiments, but it should be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were all commercially available unless otherwise specified.
Example 1
A preparation method of sargassum polysaccharide extract SP1 comprises the following operation steps:
(1) Weighing dry gulfweed powder, adding 5mL of ethanol with the volume concentration of 95% into each gram of dry gulfweed powder, refluxing for 3h at 80 ℃ until the ethanol does not change color, removing the ethanol by vacuum filtration, removing pigments in the dry gulfweed powder, drying the obtained gulfweed powder in an oven at 55 ℃, adding 15mL of distilled water into each gram of dry gulfweed powder, adding papain with the mass of 0.3% of the dry gulfweed powder, transferring the mixture into a boiling water bath at 100 ℃ after 1h in a water bath at 52 ℃ to inactivate the protease, centrifuging the mixture for 20min in the water bath at 60 ℃ for 2h and 3000r/min to obtain supernatant, repeatedly extracting precipitates once (namely adding distilled water and papain to repeat one operation), combining the two obtained supernatants, and performing rotary evaporation until the volume of the supernatant is 1/5;
(2) Adding trichloroacetic acid with the mass concentration of 25% into the substance obtained after rotary evaporation in the step (1) until the final concentration of the trichloroacetic acid is 2%, standing for 2h, centrifuging for 20min at 3000r/min, taking supernatant, concentrating, adding ethanol with the volume concentration of 95% until the final concentration of the ethanol is 80%, standing overnight, centrifuging, taking precipitate, removing protein, dissolving the precipitate with distilled water, freeze-drying, and grinding to obtain a crude sargassum polysaccharide extract;
(3) And (3) eluting and purifying 1g of the crude sargassum polysaccharide extract obtained in the step (2) by using DEAE-Sepharose Fast Flow distilled water component to obtain a sargassum polysaccharide extract distilled water component (SP 1), namely the sargassum polysaccharide extract SP1.
Example 2
A preparation method of sargassum polysaccharide extract SP1 comprises the following operation steps:
(1) Weighing dry gulfweed powder, adding 5mL of ethanol with the volume concentration of 95% into the dry gulfweed powder per gram, refluxing for 3h at 80 ℃ until the ethanol does not change color, removing the ethanol by vacuum filtration, removing pigments in the dry gulfweed powder, drying the obtained gulfweed powder in an oven at 55 ℃, adding 20mL of distilled water into the dry gulfweed powder per gram, adding papain with the mass of 0.4% of the dry gulfweed powder, transferring the powder into a boiling water bath at 100 ℃ after a water bath at 52 ℃ is carried out for 1h to inactivate the protease, centrifuging the powder for 20min at a water bath of 70 ℃ for 3h and 3000r/min to obtain supernatant, repeatedly extracting the precipitate once (namely adding distilled water and papain and repeating the operation once), combining the two obtained supernatants, and carrying out rotary evaporation until the volume of the original supernatant is 1/10;
(2) Adding trichloroacetic acid with the mass concentration of 25% into the substance obtained after rotary evaporation in the step (1) until the final concentration of the trichloroacetic acid is 2%, standing for 2h, centrifuging for 20min at 3000r/min, taking supernatant, concentrating, adding ethanol with the volume concentration of 95% until the final concentration of the ethanol is 80%, precipitating overnight, centrifuging, taking precipitate, removing protein, dissolving the precipitate with distilled water, freeze-drying, and grinding to obtain a crude sargassum polysaccharide extract;
(3) And (3) purifying 1g of the crude sargassum polysaccharide extract obtained in the step (2) by DEAE-Sepharose Fast Flow to obtain a sargassum polysaccharide extract distilled water part (SP 1), namely the sargassum polysaccharide extract SP1.
Example 3
A preparation method of sargassum polysaccharide extract SP1 comprises the following operation steps:
(1) Weighing dry gulfweed powder, adding 5mL of ethanol with the volume concentration of 95% into the dry gulfweed powder per gram, refluxing for 3h at 80 ℃ until the ethanol does not change color, removing the ethanol by vacuum filtration, removing pigments in the dry gulfweed powder, drying the obtained gulfweed powder in an oven at 55 ℃, adding 25mL of distilled water into the dry gulfweed powder per gram, adding papain with the mass of 0.5% of the dry gulfweed powder, transferring the powder into a boiling water bath at 100 ℃ after a water bath at 52 ℃ is carried out for 1h to inactivate the protease, centrifuging the powder for 20min at 80 ℃ for 4h and 3000r/min to obtain supernatant, repeatedly extracting precipitates once (namely adding distilled water and papain and repeating the operation once), combining the obtained supernatants twice, and carrying out rotary evaporation until the volume of the supernatant is 1/15;
(2) Adding trichloroacetic acid with the mass concentration of 25% into the substance obtained after rotary evaporation in the step (1) until the final concentration of the trichloroacetic acid is 5%, standing for 2h, centrifuging for 20min at 3000r/min, taking supernatant, concentrating, adding ethanol with the volume concentration of 95% until the final concentration of the ethanol is 80%, standing overnight, centrifuging, taking precipitate, removing protein, dissolving the precipitate with distilled water, freeze-drying, and grinding to obtain a crude sargassum polysaccharide extract;
(3) And (3) eluting and purifying 1g of the crude sargassum polysaccharide extract obtained in the step (2) by using DEAE-Sepharose Fast Flow distilled water component to obtain a sargassum polysaccharide extract distilled water component (SP 1), namely the sargassum polysaccharide extract SP1.
The sargassum polysaccharide obtained by the invention is almost white in color, and the obtained components have high elution rate and obvious antiviral effect.
And (3) performance testing:
test example 1 Sargassum polysaccharide physicochemical Properties
(1) Molish reaction
Preparing the sargassum polysaccharide extract SP1 prepared by the invention into a sample with the concentration of 1.0mg/mL by using distilled water, adding 1.0mL of 1mg/mL sargassum polysaccharide extract SP1 solution and 200 mu of LMolish reagent into a test tube, inclining the test tube, slowly adding 1.0mL of concentrated sulfuric acid along the tube wall, then vertically arranging the test tube, and observing the color change of the interface of the two solutions. Distilled water was used as a negative control and 0.1% starch indicator solution was used as a positive control.
TABLE 1 Molish reaction results
| Sargassum polysaccharide extract SP1 | Distilled water | Starch solution |
| Purple color appearsRing (C) | No purple ring | The purple ring appears |
(2) Iodine-potassium iodide reaction
The sargassum polysaccharide extract SP1 prepared by the invention is prepared into a sample with the concentration of 1.0mg/mL by using distilled water, 100 mu L of 1.0mg/mL sargassum polysaccharide extract SP1 solution, 100 mu L of distilled water and 100 mu L of starch with the concentration of 1.0mg/mL in each hole of a spot template are added, and finally 50 mu L of 0.1mol/L iodine solution is added in each hole to observe the reaction phenomenon. Distilled water was used as a negative control, and 1.0mg/mL starch solution was used as a control.
TABLE 2 iodine-potassium iodide reaction results (iodine solution color: brown-yellow)
| Sargassum polysaccharide extract SP1 | Distilled water | Starch solution |
| Brown yellow | Brown yellow | Blue color |
(3) Ninhydrin reaction
The sargassum polysaccharide extract SP1 prepared by the invention is prepared into a sample with the concentration of 1mg/mL by using distilled water, 1.0mL of 1mg/mL sargassum polysaccharide extract SP1 solution and 0.5mL of 0.1% ninhydrin ethanol solution are added into a test tube, mixed uniformly and boiled for 2min, then 0.5mL of glycine solution is used as a positive control, and 1.0mL of distilled water is used as a negative control. 0.5% glycine solution was used as a positive control.
TABLE 3 ninhydrin reaction results
| Group of | Color change |
| Sargassum polysaccharide extract SP1 | Unchanged and transparent |
| Glycine control group | Change from colorless to purple |
| Distilled water | No change and transparency |
Test example 2 Sargassum polysaccharide extract SP1 inhibits infection and replication of PRRSV in MARC-145 cells
1. Materials and methods
1.1 materials
The sargassum polysaccharide extract SP1 is prepared; sodium pyrophosphate purchased from Tianjin Bodi chemical corporation, production standard No.: HG3-1288-80; papain, available from Shanghai national drug group chemical reagents, production lot number: WM20070618;
the PRRSV-GXNN1396 strain (Genbank ID: MD 660067) is separated, identified and stored in a preventive veterinary medicine key laboratory of Guangxi university, and then is proliferated by MARC-145 cells; MARC-145 cell line, purchased from the cell research institute of life sciences, shanghai, chinese academy;
minimum Essential Medium (MEM culture): containing Earle's balanced salts,2.0mM L-glutamine, NEAA and Sodium Bicarbonate; when in use, 9.61g of MEM powder is weighed and dissolved in 1000mL of ultrapure water, and simultaneously 2.2g of accurately weighed NaHCO is used 3 Dissolving in the medium, shaking, mixing well to dissolve completely, filtering with 0.22 μm filter membrane for sterilization, and storing at 4 deg.C to obtain MEM culture solution;
PBS buffer solution (phosphate buffer solution) mainly containing sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride, and having pH of 7.2-7.4; PBS used in the invention is commercially available PBS powder, and the PBS slow-release solution can be obtained by dissolving the PBS powder in 2L of ultrapure water and performing high-pressure sterilization.
1.2 methods
1.2.1 antiviral infection and replication experiments
MARC-145 cell density was adjusted to 2X 10 with MEM medium containing 2% Fetal Bovine Serum (FBS) 5 mL, added to 12-well plates, 1000. Mu.L/well, placed at 37 ℃ 5% CO 2 Culturing in an incubator for about 24h to grow into a cell monolayer, then sucking and removing cell supernatant, performing experimental grouping treatment according to table 4, repeating each group for 3 times, discarding cell supernatant after 24h, washing with PBS for three times, collecting cells, and freezing and storing in a refrigerator at-80 ℃ for detection of viruses and N genes thereof.
TABLE 4 antiviral replication experiment grouping and treatment
1.2.2 antiviral Release test
Regulation of MARC-145 cell density to 2X 10 with 2% FBS-containing MEM culture 5 mL, added to 12-well plates, 1000. Mu.L/well, placed at 37 ℃ 5% CO 2 After culturing for about 24 hours in an incubator to grow an adult cell monolayer, cell supernatant was aspirated and discarded, experimental treatment was performed as shown in Table 5, corresponding viruses or drugs were added, incubation was performed for 2 hours, cell supernatant was aspirated and washed with PBS slow-release solution for 3 times, then 2% FBS-containing MEM culture medium was added, culture was continued for 48 hours, cell supernatant was collected and frozen at-80 ℃ for future use.
TABLE 5 grouping and treatment of antiviral Release experiments
2. Results
2.1 antiviral replication assay
To further determine whether the concentration of added SP1 inhibited PRRSV replication in MARC-145 cells, antiviral replication experiments were designed at different concentrations of SP1, and it can be seen from table 6 that the levels of virus replication were significantly reduced (P < 0.01) after the effect of each concentration of SP1 compared to the PRRSV control group, indicating that each concentration of SP1 (25 μ g/mL, 50 μ g/mL and 100 μ g/mL) significantly inhibited PRRSV replication in MARC-145 cells and that the number of PRRSV replication in MARC-145 cells decreased exponentially.
TABLE 6 Effect of different concentrations of SP1 treatment on PRRSV copy number in MARC-145 cells
| Group of | Viral replication index |
| Blank control group | 0±0 |
| PRRSV control group | 5.59±0.23 |
| Ribavirin control group | 3.83±0.62** |
| SP1 Low concentration group | 4.09±0.41** |
| Concentration group in SP1 | 3.73±0.43** |
| SP1 high concentration group | 4.81±0.28** |
2.2 antiviral Release assay
At 48h after virus infection, the progeny virus PRRSV has been released into the cell supernatant, and as can be seen from the data in table 7, the virus replication numbers of the cell supernatant are not statistically different (P > 0.05) in the SP1 low concentration group compared with the PRRSV control group, which indicates that the SP1 does not inhibit the PRRSV release process at the low concentration of 25 μ g/mL working concentration, whereas the SP1 50 and SP1 100 groups are significantly different (P < 0.01) from the PRRSV control group, which indicates that the SP1 at 50 μ g/mL and 100 μ g/mL can significantly inhibit the PRRSV release process, and the SP1 high concentration group is significantly different (P < 0.01) from the ribavirin control group, which indicates that the PRRSV release inhibition ability of the SP1 at the high concentration of 100 μ g/mL is better than that of ribavirin.
TABLE 7 measurement of PRRSV release levels from MARC-145 cell supernatant by different concentrations of SP1 treatment
| Group of | Numerical value of viral replication |
| Blank control group | 0±0** |
| PRRSV control group | 3.86±0.08 |
| Ribavirin control group | 3.12±0.29** |
| SP1 Low concentration group | 3.93±0.07 |
| Concentration group in SP1 | 3.11±0.21** |
| SP1 high concentration group | 1.98±0.45** ## |
In conclusion, the sargassum polysaccharide extract SP1 can effectively resist the replication and release of PRRSV in MARC-145 cells, has good antiviral effect, and is worthy of further development and utilization.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various exemplary embodiments and with various alternatives and modifications as are suited to the particular use contemplated. The scope of the invention is defined by the claims and their equivalents.
Claims (8)
1. A preparation method of sargassum polysaccharide extract SP1 is characterized by comprising the following operation steps:
(1) Weighing dry gulfweed powder, removing pigment by using ethanol, drying, and then mixing the powder according to the weight ratio of 1: 10-1: 30, adding water, then adding papain with the mass of 0.10-0.5 percent of the dry gulfweed powder, transferring the papain to a boiling water bath after the water bath to inactivate the protease, then carrying out the water bath for 1-4 h at 50-90 ℃, centrifuging to obtain a supernatant, repeatedly extracting the precipitate once, combining the supernatants obtained by the two times, and evaporating;
(2) Concentrating the substance obtained in the step (1) after evaporation, precipitating with ethanol, and freeze-drying the substance obtained after ethanol precipitation to obtain crude Sargassum polysaccharide extract;
(3) Eluting the crude sargassum polysaccharide extract by an elution column to obtain a sargassum polysaccharide extract SP1; wherein the eluting component is water.
2. The method for preparing sargassum polysaccharide extract SP1 according to claim 1, wherein: in the step (1), the pigment removal by using ethanol is to add ethanol with the volume concentration of 95% and reflux the mixture at 80 ℃ until the ethanol does not change color, and then remove the ethanol by vacuum filtration.
3. The method for preparing sargassum polysaccharide extract SP1 according to claim 1, wherein: the drying in the step (1) is drying at 55 ℃.
4. The method for preparing sargassum polysaccharide extract SP1 according to claim 1, wherein: in the step (1), after being put in a water bath at 52 ℃ for 1h, the mixture is transferred to a boiling water bath at 100 ℃ for 0.5h to inactivate the protease.
5. The method for preparing sargassum polysaccharide extract SP1 according to claim 1, wherein: and (3) the concentration and alcohol precipitation in the step (2) is to add trichloroacetic acid with the mass concentration of 25% for standing, centrifuging and concentrating in sequence, add ethanol with the volume concentration of 95% into the obtained concentrated solution for precipitating overnight, centrifuging and taking the precipitate.
6. The method for preparing sargassum polysaccharide extract SP1 according to claim 1, wherein: the elution column in the step (3) is DEAE-Sepharose Fast Flow or DEAE52.
7. Use of a sargassum polysaccharide extract SP1 prepared according to any one of claims 1 to 6 for the preparation of a medicament against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
8. A formulation against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), characterized by: the effective component of the preparation is sargassum polysaccharide extract SP1 prepared by the method of any one of claims 1-5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382200A (en) * | 2011-09-06 | 2012-03-21 | 广西大学 | Production process for separating and extracting sargasso polysaccharide by enzymatic hydrolysis method |
| CN105483183A (en) * | 2016-01-07 | 2016-04-13 | 福建农林大学 | Preparation method of sargassum oligosaccharide and application of sargassum oligosaccharide in hypoglycemic drugs |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382200A (en) * | 2011-09-06 | 2012-03-21 | 广西大学 | Production process for separating and extracting sargasso polysaccharide by enzymatic hydrolysis method |
| CN105483183A (en) * | 2016-01-07 | 2016-04-13 | 福建农林大学 | Preparation method of sargassum oligosaccharide and application of sargassum oligosaccharide in hypoglycemic drugs |
Non-Patent Citations (1)
| Title |
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| 韦英益等: "马尾藻多糖对猪脾细胞免疫活性及其抗病毒活性的影响", 《南京农业大学学报》, vol. 35, no. 2 * |
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