A diluent for Newcastle disease vaccine containing polysaccharide from American ginseng rootlets and its preparation method

Abstract

The invention relates to the technical field of biomedicine, and particularly to a diluent of a Newcastle disease vaccine containing American ginseng root polysaccharide and a preparation method thereof. American ginseng and American ginseng roots are selected for in vitro screening, and after screening, the American ginseng roots with lower cost and better effect are selected as the main component of the vaccine diluent. The American ginseng root polysaccharide is added to a phosphate buffer solution, and 1 mL of the American ginseng root polysaccharide is taken out and diluted 10 times with the phosphate buffer solution to obtain a diluent of the Newcastle disease vaccine containing American ginseng root polysaccharide. The diluent of the Newcastle disease vaccine containing American ginseng root polysaccharide having a concentration of 0.06-0.24 mg / mL of American ginseng root polysaccharide is prepared. American ginseng polysaccharide and American ginseng root polysaccharide are selected for in vitro screening, and after screening, the American ginseng root polysaccharide with lower cost and better effect is selected as the main component of the vaccine diluent. The invention solves the problem of how to use the polysaccharide in the American ginseng root for the diluent of the freeze-dried Newcastle disease vaccine of chickens to promote the proliferation of immune cells, increase the immune titer, and promote the release of cytokines.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide and a preparation method thereof. Background Art

[0002] Newcastle disease (ND) is a highly contagious viral disease caused by the Newcastle disease virus (NDV). It is highly contagious, presents diverse symptoms, and has a high mortality rate. It primarily affects poultry, particularly chickens, and has a global distribution, causing significant economic losses to the poultry industry. Vaccination is key to preventing ND, and implementing hygiene management and biosecurity measures can reduce the risk of viral transmission.

[0003] Freeze-dried attenuated vaccines are obtained through artificial attenuation or natural selection, resulting in lower toxicity and retained antigenicity. Compared to inactivated vaccines, attenuated vaccines are able to reproduce in the body, providing longer-lasting immune protection. However, they require dilution before use, typically with saline or phosphate-buffered saline (PBS). These diluents do not enhance the vaccine's protective effect but only dilute it.

[0004] American ginseng (Panax quinquefolium Linn.), also known as Western ginseng, American ginseng, and Yang ginseng, is a perennial herb belonging to the Araliaceae family. It primarily grows in North America, particularly the United States and Canada. The root of American ginseng is widely used in traditional Chinese medicine for its tonic, immune-boosting, and fatigue-fighting properties. However, the root itself is less widely used. The root itself is cheaper than the root itself.

[0005] Chinese herbal polysaccharides refer to a class of active polysaccharides extracted from Chinese herbal medicines, generally excluding starch and cellulose. These polysaccharides have multiple biological activities and are believed to maintain or enhance the body's physiological functions. Among them, studies have shown that Chinese herbal polysaccharides have immune-modulating, anti-tumor, anti-viral, hypoglycemic, and anti-aging effects. These effects have made Chinese herbal polysaccharides a focus of attention in both traditional and modern medicine, and they are widely used in the production and development of drugs, health supplements, and healthcare products. Their unique pharmacological effects provide new directions for exploration of human health and also bring new opportunities for drug research and development. Studies have found that American ginseng polysaccharides can promote immune function and have a significant inhibitory effect on the pro-inflammatory response induced by lipopolysaccharide. Therefore, American ginseng root polysaccharides can be developed as vaccine diluents with immune-enhancing effects.

[0006] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the Invention

[0007] The purpose of the present invention is to solve the problem of how to use the polysaccharide in American ginseng root hairs for the diluent of Newcastle disease freeze-dried vaccine for chickens to promote the proliferation of immune cells, improve the immune titer and promote the release of cytokines. The present invention provides a diluent of Newcastle disease vaccine for chickens containing American ginseng root hairs polysaccharide and a preparation method thereof.

[0008] In order to achieve the above object, the present invention discloses a method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide, comprising adding American ginseng root polysaccharide to a phosphate buffer solution, taking out 1 mL of the solution and diluting it 10 times with a phosphate buffer solution to obtain a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide.

[0009] The concentration of American ginseng polysaccharide in the diluent of the chicken Newcastle disease vaccine containing American ginseng polysaccharide is 0.06-0.24 mg / mL.

[0010] The preparation method of the American ginseng polysaccharide comprises the following steps:

[0011] S1, grinding the American ginseng roots into powder, mixing with RO water, and performing rotary heating extraction, followed by rotary evaporation in a water bath and vacuum concentration;

[0012] S2, adding 9 times the volume of anhydrous ethanol to the polysaccharide concentrated under negative pressure in step S1 and stirring to fully precipitate the polysaccharide, and placing the sugar-ethanol mixture in a refrigerator to precipitate for 12 hours;

[0013] S3, taking out the sugar-ethanol mixture precipitated in step S2 and centrifuging it to fully precipitate the sugar insoluble in ethanol, discarding the upper anhydrous ethanol, and drying the polysaccharide at 50° C. for 12 hours to obtain dry crude polysaccharide.

[0014] In step S1, the ratio of American ginseng roots to RO water is 1 g:10 mL.

[0015] In step S1, the rotary heating extraction temperature is 98° C. and the extraction time is 2 hours.

[0016] In step S1, the temperature of the water bath rotary evaporation is 50° C., and the time of the water bath rotary evaporation is 2 hours.

[0017] In step S1, the volume after negative pressure concentration is 50 mL.

[0018] In step S3, the centrifugal speed is 4000 rpm and the centrifugal time is 20 min.

[0019] The invention also discloses a diluent of a chicken Newcastle disease vaccine containing American ginseng polysaccharide prepared by the preparation method.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses American ginseng root polysaccharide for Newcastle disease freeze-dried vaccine diluent, and has the effects of promoting immune cell proliferation, improving immune titer, and promoting the release of cytokines, which can greatly improve the immune effect of the Newcastle disease vaccine, quickly produce antibodies and cytokines, and improve the body's ability to resist diseases. The American ginseng root part is selected, the raw material price is low (compared with American ginseng decoction pieces), the extraction method is simple, and the selected American ginseng polysaccharide has a wide safe concentration and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The in vitro lymphocyte proliferation effect of American ginseng root polysaccharide;

[0022] Figure 2 The in vitro lymphocyte proliferation effect of American ginseng polysaccharide;

[0023] Figure 3 This is the result of the hemagglutination inhibition test in the high-dose group of American ginseng polysaccharide;

[0024] Figure 4 This is the result of the hemagglutination inhibition test in the medium-dose group of American ginseng polysaccharide;

[0025] Figure 5 This is the result of the hemagglutination inhibition test in the low-dose group of American ginseng polysaccharide;

[0026] Figure 6 The effect of high dose of American ginseng polysaccharide on chicken cytokines;

[0027] Figure 7 The effect of medium dose of American ginseng polysaccharide on chicken cytokines;

[0028] Figure 8 The effect of low-dose American ginseng polysaccharide on chicken cytokines. DETAILED DESCRIPTION

[0029] The above and other technical features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.

[0030] Example 1

[0031] Preparation of polysaccharides

[0032] 50 g of American ginseng and 500 mL of American ginseng root were ground into a powder, mixed with 500 mL of RO water, and extracted by rotary heating at 98°C for 2 h. The mixture was then rotary evaporated in a 50°C water bath for 2 h and concentrated to 50 mL under negative pressure. Nine volumes of anhydrous ethanol (450 mL) were added and stirred to fully precipitate the polysaccharides. The sugar-ethanol mixture was refrigerated at 4°C for 12 h. After removal, it was centrifuged at 4000 rpm for 20 min to fully precipitate the ethanol-insoluble sugars, and the supernatant ethanol was discarded. The polysaccharides were dried at 50°C for 12 h to obtain dry crude polysaccharides. After grinding, the mixture was stored in a desiccator. Sugar content was determined using the phenol-sulfuric acid method. Water content was determined by loss on drying, and total saponin content was determined by the vanillin-acetic acid method. The results are shown in Table 1.

[0033] Table 1 Test results of ingredients of American ginseng roots and American ginseng slices

[0034] Moisture content Total saponin content Polysaccharide extraction rate Polysaccharide content Cost per gram of sugar American ginseng roots 11.90％ 7.10％ 15.10％ 81.30％ 0.7 yuan American ginseng slices 11.00％ 11.80％ 5.30％ 43％ 7.1 yuan

[0035] Example 2

[0036] In vitro activity test of polysaccharides

[0037] 60-day-old laying roosters were sacrificed by bleeding from the jugular vein. The spleen was removed aseptically, washed three times with Hanks solution, chopped into pieces in a 5 mL Hanks solution dish, passed through a 0.45 μm cell sieve, and the cell fluid was collected. Lymphocyte separation solution was added, centrifuged at 2000 rpm for 10 min, washed twice with Hanks solution, and the cells were counted using a cell counting plate. The cell concentration was adjusted to 1 × 10 7 / mL. The calculation formula is as follows:

[0038]

[0039] 100 μL was added to each well of a 96-well cell culture plate, and then 100 μL of 2-fold gradient dilutions of American ginseng polysaccharides and polysaccharides from American ginseng roots filtered through a 0.22 μm filter membrane were added, and each concentration was repeated in 6 wells. The cell plate was placed in a 37°C, 5% CO2 incubator for 44 hours, and then 20 μL of MTT (5 mg / mL) was added to each well. After further incubation for 4 hours, the plate was taken out, centrifuged at 2500 rpm for 10 minutes, 100 L was removed, and 100 μL of DMSO was added to each well to dissolve the precipitate. The absorbance at 570 nm (A) was measured by enzyme-linked immunosorbent assay. 570 value), as an indicator of lymphocyte proliferation, the results are as follows Figure 1 、 2The figure shows the in vitro activity test of American ginseng polysaccharides, which shows that American ginseng polysaccharides at 1000μg / mL to 250μg / mL and polysaccharides in American ginseng roots at 3.7411μg / mL to 0.4157μg / mL have a significant promoting effect on the proliferation of chicken spleen lymphocytes (P<0.01). This shows that both American ginseng polysaccharides and polysaccharides in American ginseng roots have immune promoting activity in vitro, but the activity of polysaccharides in American ginseng roots is stronger than that of American ginseng polysaccharides and the cost is low. Therefore, American ginseng roots were selected as the main raw material for vaccine diluent.

[0040] Example 3

[0041] Preparation of vaccine diluent

[0042] Diluted with 0 mg / mL, 0.06 mg / mL, 0.12 mg / mL, and 0.24 mg / mL of American ginseng polysaccharide respectively.

[0043] a. Add 6 mg of American ginseng polysaccharide to 10 mL of phosphate buffer, then take out 1 mL and dilute it 10-fold with phosphate buffer to obtain an animal vaccine diluent with an American ginseng polysaccharide concentration of 60 μg / mL. American ginseng polysaccharide was prepared according to Example 1.

[0044] b. Add 12 mg of American ginseng polysaccharide to 10 mL of phosphate buffer, and then take out 1 mL and dilute it 10 times with phosphate buffer to obtain an animal vaccine diluent with an American ginseng polysaccharide concentration of 120 μg / mL. American ginseng polysaccharide was prepared according to Example 1.

[0045] 24 mg of American ginseng polysaccharide was added to 10 mL of phosphate buffer, and 1 mL was taken out and diluted 10 times with phosphate buffer to obtain an animal vaccine diluent with an American ginseng polysaccharide concentration of 240 μg / mL. American ginseng polysaccharide was prepared according to Example 1.

[0046] Example 4

[0047] Immunity test of American ginseng polysaccharide vaccine diluent on Newcastle disease vaccine in chickens

[0048] Newcastle disease vaccine (La Sota strain, batch number: 202409) was purchased from Qingdao Lijian Biotechnology Co., Ltd. The vaccine was diluted and shaken well with the above-prepared vaccine diluent.

[0049] One hundred 14-day-old Dawu Jinfeng laying hens were randomly divided into five groups, with 20 chickens in each group: a high-dose American ginseng polysaccharide group (AGPH), a medium-dose group (AGPM), a low-dose group (AGPL), a vaccine control group (VC), and a blank control group (BC). Except for the blank control group (normal saline was administered intranasally and intraocularly), the remaining four groups were vaccinated with a Newcastle disease vaccine diluted with American ginseng polysaccharide at concentrations of 0 mg / mL, 0.06 mg / mL, 0.12 mg / mL, and 0.24 mg / mL, respectively, via intranasal and intraocular administration. A secondary immunization was performed 14 days after immunization. Blood was collected from the subwing vein at 7, 14, 21, 28, 35, and 42 days after immunization. Serum was isolated, and antibody titers were measured using hemagglutination (HA) and hemagglutination inhibition (HI) assays.

[0050] Example 5

[0051] Antibody titer detection

[0052] 1. Preparation of 1% chicken red blood cell suspension

[0053] Collect 3 mL of blood from the subwing vein of a specific pathogen-free rooster. Add 3 volumes of normal saline, mix thoroughly, and centrifuge at 1000 rpm for 8 minutes at 4°C. Discard the supernatant. Add 3 volumes of normal saline and repeat the above washing process three times. Pipette 500 μL of the red blood cell pellet into a sterile centrifuge tube containing 45.5 mL of PBS to prepare a red blood cell suspension. Gently shake the centrifuge tube to resuspend the red blood cells.

[0054] 2. Add 25 μL of PBS to each well of a conical-bottom 96-well plate. Add 25 μL of antigen to the first well, and dilute it serially to the 11th well. Discard 25 μL of the liquid in the 11th well, and then add 25 μL of 1% chicken red blood cell suspension to each well. Let it stand for 30 minutes before determining the results.

[0055] Results determination: Tilt the conical-bottom 96-well plate at a certain angle and observe the red blood cell pellet. If the pellet quickly flows down in a linear pattern, it indicates that the viral antigen has not or incompletely agglutinated with the red blood cells. If the red blood cell pellet does not flow down or does not form a pellet, but is distributed in a thin layer, it indicates that the viral antigen has agglutinated with the red blood cells. Generally, the dilution factor of the first well in which the pellet flows down in a linear pattern is used as the highest dilution factor of the test antigen, which is the antigen titer.

[0056] 3. Verification of Four-Unit Antigen

[0057] Prepare a four-unit antigen using PBS based on the HA test results. Add 25 μL of PBS to wells 1 to 5 of a conical-bottom 96-well plate. Add 25 μL of the prepared four-unit antigen to well 1, mix thoroughly, and then dilute the mixture serially to well 5. Discard 25 μL of the mixture and add 25 μL of a 1% chicken red blood cell suspension to each well. Let stand for 30 minutes and observe the results. Interpret the results: Complete aggregation in the first two wells, half of the red blood cells in well 3 immediately become streamlined, and all of the red blood cells in wells 4 and 5 immediately become streamlined. This indicates that the four-unit antigen is accurate and the verification is successful.

[0058] 4. Hemagglutination inhibition test (HI)

[0059] Add 25 μL of PBS to each well of a conical-bottom 96-well plate, add 25 μL of the test serum to the first well, dilute serially to the 11th well, discard 25 μL of liquid from the 11th well, add 25 μL of four units of antigen to each well, let it react for 30 minutes, then add 25 μL of 1% chicken red blood cell suspension to each well, let it react for 30 minutes, and observe the results.

[0060] Read the results: The maximum dilution that makes the red blood cell sediment flow linearly is the hemagglutination inhibition (HI) titer of the serum sample, expressed as Log2. The result is as follows: Figure 3 、 4 , as shown in 5.

[0061] Statistical analysis

[0062] One-way ANOVA was performed using Graphpad Pism 9.5 software. All data were expressed as mean ± standard deviation. P>0.05 indicated no significant difference, P<0.05 indicated a significant difference, and P<0.01 indicated an extremely significant difference.

[0063] like Figure 3 As shown in the results, after the first immunization, the NDV-specific antibody level in the high-dose American ginseng polysaccharide (AGPH) group increased. The NDV-specific antibody level in the AGPH group was significantly higher than that in the control group 7 and 14 days after the first immunization (P < 0.05) and 7 and 14 days after the second immunization.

[0064] like Figure 4 As shown in the data, it can be concluded that after the first immunization, the NDV-specific antibody levels in the chicken serum of the immunized group and the medium-dose American ginseng polysaccharide (AGPM) group increased. The NDV-specific antibody levels in the AGPM group were higher than those in the immunized control group 7 and 14 days after the first immunization (P < 0.05) and 7 and 14 days after the second immunization.

[0065] like Figure 5As shown in the data, it can be concluded that after the first immunization, the NDV-specific antibody levels in the chicken serum of the immunized group and the low-dose American ginseng polysaccharide (AGPL) group increased. The NDV-specific antibody levels in the AGPL group were higher than those in the immunized control group 7 and 14 days after the first immunization (P < 0.05) and 7 and 14 days after the second immunization.

[0066] The serum antibody levels of the American ginseng polysaccharide group were significantly higher than those of the blank control group and the vaccine control group on days 14, 21, 28, and 35 after immunization (P<0.05). The American ginseng polysaccharide medium-dose group showed better immune effects on days 28, 35, and 42.

[0067] Example 6

[0068] Effects of American ginseng polysaccharide vaccine diluent on chicken cytokines

[0069] One week after the second immunization, the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method was used to detect IL-2, IL-4, IL-6, and INF-γ in the experimental chickens.

[0070] Set up standard wells, 0 value wells, blank wells and sample wells. Add 50 μL of standard of different concentrations to each standard well, add 50 μL of sample diluent to the 0 value wells, add nothing to the blank wells, and add 50 μL of the sample to be tested to the sample wells.

[0071] In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to the standard wells, 0 value wells, and sample wells.

[0072] Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator in the dark for 60 minutes.

[0073] Remove the sealing film, discard the liquid, and pat dry on absorbent paper. Fill each well with wash solution, let it sit for 20 seconds, shake off the wash solution, and pat dry on absorbent paper. Repeat this process five times. After washing, pat the reaction plate dry on clean, non-linting paper before adding substrate.

[0074] Thoroughly mix substrates A and B at a 1:1 volume ratio and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with sealing film and incubate in a 37°C water bath or incubator in the dark for 15 minutes.

[0075] Add 50 μL of stop solution to all wells, and read the absorbance (OD value) of each well on a microplate reader.

[0076] With the concentration of the standard as the horizontal axis (6 standard wells, plus 1 0 value well, a total of 7 concentration points), and the corresponding absorbance (OD value) as the vertical axis, computer software is used to fit the four-parameter logistic curve (4-pl) to create a standard curve equation. The concentration value of the sample is calculated using the equation through the absorbance (OD value) of the sample. The measured value is as follows Figure 5 、 6 , as shown in 7.

[0077] like Figure 6 As shown in the figure, it can be concluded that the IL-2, IL-4, IL-6, and INF-γ levels of chickens after vaccination with the American ginseng polysaccharide vaccine diluent were significantly higher (P < 0.01) than those in the ordinary vaccine group and the blank control group, indicating that high-dose American ginseng polysaccharides can promote the production of IL-2, IL-4, IL-6, and INF-γ in chicken cells.

[0078] like Figure 7 As shown in the figure, it can be concluded that the IL-2, IL-4, IL-6, and INF-γ levels of chickens after vaccination with the American ginseng polysaccharide vaccine diluent were significantly higher (P < 0.01) than those in the ordinary vaccine group and the blank control group, indicating that a medium dose of American ginseng polysaccharide can promote the production of IL-2, IL-4, IL-6, and INF-γ in chicken cells.

[0079] like Figure 8 As shown in the results, it can be concluded that the IL-2, IL-4, IL-6, and INF-γ levels of chickens after vaccination with the American ginseng polysaccharide vaccine diluent were significantly (P < 0.01) higher than those in the ordinary vaccine group and the blank control group, indicating that low-dose American ginseng polysaccharides can promote the production of IL-2, IL-4, IL-6, and INF-γ in chicken cells.

[0080] The above description is merely a preferred embodiment of the present invention and is intended to be illustrative rather than restrictive of the present invention. Those skilled in the art will appreciate that many changes, modifications, and even equivalents may be made to the present invention within the spirit and scope of the claims, all of which fall within the scope of protection of the present invention.

Claims

1. A method for preparing a diluent of a Newcastle disease vaccine containing American ginseng root polysaccharide, characterized in that: Adding American ginseng root polysaccharide to phosphate buffer, taking out 1 mL and diluting 10 times with phosphate buffer to obtain a dilution of chicken Newcastle disease vaccine containing American ginseng root polysaccharide, wherein the concentration of American ginseng root polysaccharide in the dilution of chicken Newcastle disease vaccine containing American ginseng root polysaccharide is 0.12 mg / mL; The preparation method of the American ginseng root polysaccharide comprises the following steps: S1, grinding the American ginseng roots into powder, mixing with RO water, and performing rotary heating extraction, followed by rotary evaporation in a water bath and vacuum concentration; S2, adding 9 times the volume of anhydrous ethanol to the polysaccharide concentrated under negative pressure in step S1 and stirring to fully precipitate the polysaccharide, and placing the sugar-ethanol mixture in a refrigerator to precipitate for 12 hours; S3, taking out the sugar-ethanol mixture precipitated in step S2 and centrifuging it to fully precipitate the sugar insoluble in ethanol, discarding the upper layer of anhydrous ethanol, and drying the polysaccharide at 50° C. for 12 hours to obtain dried American ginseng root polysaccharide.

2. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, wherein: In step S1, the ratio of American ginseng roots to RO water is 1 g:10 mL.

3. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, wherein: In step S1, the rotary heating extraction temperature is 98° C. and the extraction time is 2 hours.

4. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, wherein: In step S1, the temperature of the water bath rotary evaporation is 50° C., and the time of the water bath rotary evaporation is 2 hours.

5. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, wherein: In step S1, the volume after negative pressure concentration is 50 mL.

6. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, wherein: In step S3, the centrifugal speed is 4000 rpm and the centrifugal time is 20 min.

7. A diluent of a Newcastle disease vaccine containing American ginseng root polysaccharide prepared by the preparation method according to any one of claims 1 to 6.

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