Drug extracts for enhancing the efficacy of porcine reproductive and respiratory syndrome vaccines, their preparation methods and applications

Abstract

This invention discloses a drug extract for enhancing the efficacy of porcine reproductive and respiratory syndrome (PRRS) vaccines, its preparation method, and its application. The drug extract comprises aqueous extracts of Astragalus membranaceus and Curcuma zedoaria, wherein the optimal mass ratio of Astragalus membranaceus to Curcuma zedoaria is 4-5:1. The Astragalus membranaceus and Curcuma zedoaria extract of this invention enhances the efficacy of existing PRRSV vaccines, improves vaccine resistance to PRRSV, and effectively stimulates the body to produce sufficient neutralizing antibodies, thereby enhancing the overall immune effect of the vaccine, with significantly better results than using the vaccine alone.

Description

Technical Field

[0001] This invention relates to a pharmaceutical extract, specifically to a pharmaceutical extract for enhancing the efficacy of a porcine reproductive and respiratory syndrome (PRRS) vaccine, its preparation method, and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes severe reproductive disorders, growth retardation, respiratory symptoms, and high mortality in pigs. Furthermore, the disease primarily causes immunosuppression, leading to decreased immunity and secondary infections by other pathogens. Clinically, it is extremely difficult to control and has a very high mortality rate. It is listed as a notified disease by the World Organisation for Animal Health (OIE), and in my country, it is classified as a Class II animal disease. Currently, HP-PRRSV (highpathogenicity-PRRSV), NADC34-like PRRSV, and NADC30-like PRRSV strains coexist in my country, and new recombinant variants are constantly emerging. However, HP-PRRSV remains the most serious threat to the pig industry. HP-PRRSV is characterized by a 30 (29+1) amino acid deletion in the NSP2 gene. It can cause morbidity rates of up to 100% and mortality rates of over 50% in piglets, abortion rates of over 30% in sows, and can also cause disease and death in fattening pigs. Representative strains include JXA1 (Genbank ID: EF112445), TJ (Genbank ID: EU860248), and HuN4 (Genbank ID: EF635006).

[0003] Astragalus is the dried root of Astragalus mongholicus or Astragalus membranaceus, both belonging to the legume family. Astragalus has the effects of strengthening the spleen and replenishing the middle jiao, raising yang and lifting prolapse, benefiting wei qi and consolidating the exterior, promoting diuresis, and promoting tissue regeneration and detoxification. It is mainly used to treat spleen qi deficiency, lung qi deficiency, spontaneous sweating due to qi deficiency, qi and blood deficiency, and sores that are difficult to ulcerate or heal.

[0004] Curcuma zedoaria is the dried rhizome of *Curcuma zedoaria*, *Curcuma guangxiong*, or *Curcuma wenyujin*, all belonging to the ginger family. The term "Curcuma zedoaria" was first recorded in the *Treatise on the Nature of Medicinal Herbs*: "It grows abundantly in the western regions and southern provinces. Its leaves resemble lotus leaves, and its seeds resemble dried chess pieces. It grows abundantly beneath the roots. It has two properties: good and bad. The bad one is poisonous." Curcuma zedoaria is bitter and pungent, warm in nature, and non-toxic. According to the *Compendium of Materia Medica*, "Curcuma zedoaria, when roasted with *Saussurea costus*, treats all kinds of cold-induced sharp pain in the heart, which can be fatal. It is also used for chronic abdominal pain that recurs intermittently." Modern research has found that its main component is volatile oil.

[0005] The combination of Astragalus membranaceus and Curcuma zedoaria originates from the Li Chong Tang formula in Zhang Xichun's *Medical Records of Integrating Chinese and Western Medicine*. This formula primarily treats qi deficiency and blood stasis syndrome. Curcuma zedoaria dispels blood stasis in the Chong meridian, while Astragalus membranaceus protects qi and blood, thus removing blood stasis without damaging qi and blood. Furthermore, Astragalus membranaceus tonifies qi, and with the aid of Curcuma zedoaria to promote circulation, the tonification is not stagnant, and the original qi becomes stronger. With stronger original qi, the power of Curcuma zedoaria is further enhanced to eliminate masses, hence its effectiveness. Traditional Chinese medicine believes that the pathogenesis of liver cancer is mostly due to deficiency of vital qi and stagnation of blood stasis. Astragalus membranaceus tonifies vital qi, while Curcuma zedoaria breaks up blood stasis and eliminates masses. The combination of the two not only complements each other's weaknesses but also leverages their respective advantages, achieving the effect of breaking up blood stasis without harming vital qi and promoting qi circulation without leaving stagnation.

[0006] Some commonly used PRRSV vaccines still have shortcomings. Taking the PRRSV modified-live virus (MLV) vaccine as an example, the main advantage of an MLV vaccine is its ability to induce a protective immune response similar to PRRSV infection. Ideally, an MLV vaccine should have heterologous protection. However, MLV vaccines do not provide strong protection in animals and cannot combat genetically different wild-type strains. Furthermore, the biggest problem with any MLV vaccine is its ability to restore virulence. The continuous evolution of the PRRSV virus and the emergence of more pathogenic strains in recent years have presented ongoing challenges to the development of next-generation MLV vaccines. Inactivated vaccines also have many problems, such as low immunogenicity, poor immunogenicity in evoking neutralizing antibody immunity, and cumbersome immunization processes. In addition to optimizing next-generation PRRSV vaccines using existing biotechnology, it is also essential to develop novel vaccine synergists for use in combination with vaccines, utilizing relevant theories from Traditional Chinese Medicine research. Summary of the Invention

[0007] The purpose of this invention is to provide a drug extract for enhancing the efficacy of porcine reproductive and respiratory syndrome (PRRS) vaccines, its preparation method, and its application. This extract enhances the efficacy of existing PRRSV vaccines, improves the resistance of vaccines to them, and can effectively stimulate the body to produce sufficient neutralizing antibodies, thereby enhancing the overall immune effect of the vaccine. The effect is significantly better than using the vaccine alone.

[0008] To achieve the above objectives, the present invention provides a drug extract for enhancing the efficacy of a porcine reproductive and respiratory syndrome (PRRS) vaccine. The drug extract comprises an aqueous extract of Astragalus membranaceus and Curcuma zedoaria, wherein the mass ratio of Astragalus membranaceus to Curcuma zedoaria is 2 to 5:1.

[0009] Preferably, the mass ratio of Astragalus membranaceus to Curcuma zedoaria is 4-5:1.

[0010] Another object of the present invention is to provide the application of the aforementioned drug extract in enhancing the efficacy of porcine reproductive and respiratory syndrome (PRRS) vaccines.

[0011] Preferably, the drug extract is capable of enhancing the ability to inhibit the virus after administration of a porcine reproductive and respiratory syndrome vaccine.

[0012] Preferably, the drug extract can significantly downregulate the mRNA levels of IL-β and TNF-α and upregulate the mRNA level of IFN-α after administration of porcine reproductive and respiratory syndrome vaccine.

[0013] Preferably, the concentration of the drug extract is 50–60 μg / mL.

[0014] Another object of the present invention is to provide a method for preparing the aforementioned drug extract, the method comprising: decocting Astragalus membranaceus and Curcuma zedoaria in water at a mass ratio of 2 to 5:1 over a low heat several times; collecting the filtrate, concentrating it, drying the resulting extract, granulating it, and obtaining the Astragalus membranaceus and Curcuma zedoaria extract.

[0015] Preferably, the food is soaked before the first decocting.

[0016] Preferably, the decoction is performed three times over a low flame, and the ratio of the total mass of Astragalus membranaceus and Curcuma zedoaria to the volume of water added in the first, second, and third decoctions is 1g: 13-14mL: 10-11mL: 7-8mL.

[0017] Preferably, the first, second, and third decocting times are 60 min, 45 min, and 30 min, respectively.

[0018] The drug extract for enhancing the efficacy of porcine reproductive and respiratory syndrome (PRRS) vaccine of the present invention, its preparation method, and its application have the following advantages:

[0019] (1) The Astragalus and Curcuma extract of the present invention enhances the efficacy of existing PRRSV vaccines, especially for wild strains with high genetic diversity and rapidly mutating strains, thereby increasing the vaccine’s resistance to them.

[0020] (2) The Astragalus and Curcuma extract of the present invention can effectively stimulate the body to produce sufficient neutralizing antibodies for inactivated vaccines with low immunogenicity, thereby improving the overall immune effect of the vaccine and the effect is significantly better than that of using the vaccine alone.

[0021] (3) The Astragalus and Curcuma extract of the present invention has low cost of Astragalus and Curcuma slices, and the extraction process is simple and easy to operate, making it suitable for promotion in production;

[0022] (4) The Astragalus and Curcuma extract of the present invention promotes the sustainable development of the aquaculture industry. By providing a more effective and safer PRRSV vaccine enhancement strategy, it helps to reduce the losses caused by PRRSV in the aquaculture industry and promotes the healthy and sustainable development of the aquaculture industry. Attached Figure Description

[0023] Figure 1 This is a photograph showing the appearance of the Astragalus and Curcuma zedoaria extract of the present invention.

[0024] Figure 2 The survival rate of Pams-163 of the present invention under the action of Astragalus and Curcuma zedoaria extracts in different ratios and concentrations.

[0025] Figure 3 The results of different formulations of Astragalus membranaceus and Curcuma zedoaria extracts with different action modes of PRRSV are shown in the figure; the viral titer is presented as mean ± variance (3 replicates per sample); VC represents the viral control group.

[0026] Figure 4 This is the result of RT-qPCR detection of the relative expression levels of cytokine mRNA after the extract inhibited the virus, according to the present invention; in the figure, C:A represents Curcuma zedoaria and Astragalus membranaceus.

[0027] Figure 5 The survival rate of Pams-163 of the present invention under the action of Astragalus membranaceus extract and Curcuma zedoaria extract, respectively.

[0028] Figure 6 This invention presents the results of different mechanisms of action of Astragalus membranaceus extract and Curcuma zedoaria extract against PRRSV.

[0029] Figure 7 This invention uses RT-qPCR to detect the relative expression levels of cytokine mRNA after two extracts individually inhibited the virus.

[0030] Figure 8 Clinical symptom observation after PRRSV vaccination for this invention; wherein, A: clinical symptom score; B: rectal temperature statistics; C: average daily weight gain statistics.

[0031] Figure 9 This is the statistical result of the S / P value of the PRRSVN protein antibody level in this invention.

[0032] Figure 10 The results are statistical results of cytokines in piglets according to the present invention.

[0033] Note: The significance of the difference was analyzed using Pearson's r2. Compared with the virus control group, the difference was not significant: ns (p>0.05); significant: * / ^ / + (p<0.05); highly significant: ** / ^^ / ++ (p<0.01); extremely significant: *** / ^^^ / +++ (p<0.001); extremely significant: **** / ^^^^ / ++++ (p<0.0001). Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The materials used in the following experimental examples are as follows:

[0036] Astragalus and Curcuma zedoaria slices were purchased from Weikang Pharmaceutical Chain Co., Ltd. in Hongya County, Sichuan Province.

[0037] Porcine alveolar macrophages-163 (Pams-163) were preserved and provided by the Animal Disease Laboratory of Sichuan Agricultural University; PRRSV strain SCCD22 (belonging to the PRRSV NADC30-like strain, a recombinant variant strain) was preserved and provided by the Animal Disease Laboratory of Sichuan Agricultural University.

[0038] RPMI 1640 medium, fetal bovine serum, and 0.25% trypsin solution were purchased from Gibco, USA; AGRNAexPro RNA extraction reagent, SYBR Green Pro Taq HS premixed qPCR kit, and Evo M-MLVRT Kit reverse transcription kit were purchased from Hunan Aikerui Biotechnology Co., Ltd.; and the CCK-8 kit was purchased from Beyotime Biotechnology Co., Ltd.

[0039] All statistical analyses in the following experiments were performed using one-way ANOVA in SPSS 18.0. Data with p < 0.05 were selected as the criterion for statistical significance.

[0040] Experimental Example 1: Proliferation and Titer Determination of PRRSV SCCD22

[0041] 1. Cell inoculation

[0042] Pams-163 cells were cultured in RPMI 1640 medium. When the cells in the T25 cell flask reached 80-90% confluence, the medium was discarded, the cells were washed three times with PBS, and PRRSV virus solution was added. The cells were incubated at 37°C for 1.5 hours, with the flask shaken every 20 minutes to ensure even distribution of the virus solution. After the adsorption was complete, the virus solution was discarded, and 2% FBS medium was added. The cells were then placed in a 5% CO2 incubator and cultured at 37°C. The cytopathic effect was observed daily. When the cytopathic effect reached 80%, the cells were subjected to three freeze-thaw cycles, centrifuged at 5000 rpm for 10 minutes, filtered through a 0.22 μm filter, and the virus solution was aliquoted and stored at -80°C.

[0043] 2. Virus titer determination

[0044] Pams-163 cells were digested and passaged into 96-well plates. When cell confluence reached 80-90%, the purified virus solution was serially diluted 10-fold with maintenance medium (RPMI 1640 medium containing 2% FBS). -1 ~10 -9 ), 100 μL / well, 8 replicates per dilution, negative control with 100 μL maintenance medium (containing 2% FBS), incubated at 37℃ in a 5% CO2 cell culture incubator for 72–120 h, CPE observed and recorded daily, TCID of PRRSV isolates calculated according to the Reed-Muench method. 50 The results are shown in Table 1. The MOI (Multiple of Infection) was used to calculate the infection multiplicity on the cells. The MOI was used in subsequent experiments to ensure consistent experimental conditions.

[0045] Table 1PRRSV SCCD22TCID 50 Measurement results

[0046]

[0047]

[0048] Experimental Example 2: Screening of the in vitro ratio of Astragalus membranaceus and Curcuma zedoaria

[0049] 1. Preparation of water extracts of Astragalus membranaceus and Curcuma zedoaria

[0050] Astragalus and Curcuma zedoaria slices (total weight 100g) were decocted in water at a mass ratio of Astragalus:Curcuma zedoaria = 2-5:1. The specific method was as follows: Astragalus and Curcuma zedoaria were weighed and prepared according to the ratio, placed in a 3000mL beaker, and decocted three times over low heat. The amount of distilled water added each time was 1300mL, 1040mL, and 780mL, respectively. The decoction time was 60min, 45min, and 30min, respectively. The first decoction was pre-soaked for 2 hours. The filtrates were collected three times, allowed to stand for 2 hours, and then filtered again to obtain approximately 1300mL of decoction. The decoction was poured into a round-bottom flask and concentrated to 50mL at low temperature using a rotary evaporator. The weight was measured, the relative density was calculated, and the solution was stored in a refrigerator for later use. The resulting extract was dried, granulated, and obtained astragalus and curcuma zedoaria extract. (See [link to relevant documentation]). Figure 1 Astragalus and Curcuma zedoaria extract is a yellow powder.

[0051] 2. CCK-8 assay to detect the effect of different concentrations of Astragalus membranaceus and Curcuma zedoaria extract on Pams-163.

[0052] Pams-163 cells were seeded in 96-well plates and, when they reached approximately 90% confluence, the cell culture medium was discarded, and the cells were washed three times with PBS. Different concentrations of Astragalus membranaceus and Curcuma zedoaria extract were prepared by serially diluting the extract 2-fold with cell maintenance medium, resulting in concentrations of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 60 μg / mL, 30 μg / mL, 15 μg / mL, and 7.5 μg / mL. 100 μL of the cell maintenance medium containing the Astragalus membranaceus and Curcuma zedoaria extract was added to each well of a 96-well plate containing a monolayer of Pams-163 cells, with six replicates for each concentration. A cell control (cells + culture medium + CCK8 solution) and a control without the extract (culture medium + CCK8) were also included. After incubation at 37°C for 48 h, CCK-8 reagent (10 μL / well) was added. After incubation at 37°C for 1 h, the OD value was measured at 450 nm using a microplate reader. Calculate the cell survival rate after treatment with different concentrations of extract.

[0053] Cell viability (%) = (OD value of experimental group - OD value of blank group well) / (OD value of control well - OD value of blank well) × 100%.

[0054] The results are as follows Figure 2 As shown, when the concentration of Astragalus and Curcuma zedoaria extracts with different ratios is ≤60μg / mL, the survival rate of Pams-163 is ≥0.9. Therefore, the maximum safe concentration of Astragalus and Curcuma zedoaria extracts with different ratios for Pams-163 is 60μg / mL.

[0055] 3. Screening of the mechanism of action of Astragalus membranaceus and Curcuma zedoaria extracts against PRRSV

[0056] PAMs-163 cells were seeded in 6-well plates and cultured in RPMI 1640 medium containing 10% FBS until the cells reached 80% confluence. The following groups were established: a virus infection group (PRRSV treated alone), a direct drug action group (drug and virus mixed in equal proportions), a drug prevention group (drug first, then virus), a drug inhibition group (virus first, then drug), an adsorption group (adsorption at 4℃), and a blank control group. Different concentration gradients of Astragalus and Curcuma zedoaria extracts were used, at ratios of 60 μg / mL, 30 μg / mL, 15 μg / mL, and 7.5 μg / mL.

[0057] The specific processing method is as follows:

[0058] Virus infection group – Discard cell culture medium, inoculate each well with 400 μL of PRRSV virus solution with MOI = 0.1, add 1.3 mL of maintenance medium, mix well, and incubate at 37℃ with 5% CO2.

[0059] In the direct drug action group—the cell culture medium was discarded. Based on the safe concentration of the extract, different ratios of Astragalus membranaceus and Curcuma zedoaria extracts were mixed with virus solution of MOI=0.1 at a ratio of 1:1. 400 μL of each extract was inoculated into each well, followed by 1.3 mL of maintenance medium. The mixture was then mixed and incubated at 37°C with 5% CO2.

[0060] Drug prevention group – Discard cell culture medium. Based on the safe concentration of extract, different ratios of Astragalus membranaceus and Curcuma zedoaria extract were first inoculated into 6-well plates, 400 μL per well. After incubation for 4 h, the extract was aspirated. 400 μL of PRRSV virus solution with MOI = 0.1 was added to each well. After incubation at 37℃ and 5% CO2 for 1.5 h, the virus solution was discarded. 1.7 mL of maintenance medium was added to each well and cultured at 37℃ and 5% CO2.

[0061] Drug inhibition group – Discard the cell culture medium, inoculate each well of a 6-well plate with 400 μL of PRRSV virus solution with MOI = 0.1, incubate at 37°C and 5% CO2 for 1.5 h, then discard the virus solution. Then, based on the safe concentration of the extract, add different ratios of Astragalus and Curcuma extract, 400 μL per well, along with 1.3 mL of maintenance medium, and incubate at 37°C and 5% CO2.

[0062] Adsorption group – cell culture medium was discarded. Based on the safe concentration of the extract, different ratios of Astragalus membranaceus and Curcuma zedoaria extracts were mixed with virus solution of MOI=0.1 at a ratio of 1:1. 400 μL was inoculated into each well of a 6-well plate and incubated at 4℃ for 1.5 h for adsorption. The virus solution was then discarded, and 1.3 mL of maintenance medium was added to each well. The plate was then incubated at 37℃ with 5% CO2 for further incubation.

[0063] Blank control group – Discard the cell culture medium, add 1.7 mL of maintenance medium, mix well, and incubate at 37℃ with 5% CO2.

[0064] After 24 hours of culture, total RNA was extracted from each well of cells, and the viral copy number was calculated using RTqPCR to determine the mechanism of action of the extract on the virus.

[0065] The results are as follows Figure 3 As shown, adsorption at all formulations failed to inhibit the PRRSV viral copy number (P > 0.05). Comparing the other three modes of action: all extracts at a concentration of 60 μg / mL maximally reduced the PRRSV copy number; the 60 μg / mL Astragalus and Curcuma zedoaria extract resulted in the lowest viral copy number (0.21–1.37 × 10⁻⁶). 4 The most significant decrease in viral copy number was observed at the concentration of [number of copies / mL] (p < 0.0001), which was superior to the viral copy number reduction under prophylactic effects at the same concentration (11.17–31.99 × 10⁻⁶ copies / mL). 4 The number of viral copies / mL and the number of viral copies under direct exposure (12.93–26.32 × 10⁻⁶) were also mentioned. 4 The results showed that the best mechanism of action of Astragalus membranaceus and Curcuma zedoaria extract against PRRSV was inhibition. Furthermore, in the inhibitory effect, the viral load was significantly lower when the ratio of Curcuma zedoaria to Astragalus membranaceus was 1:4 and 1:5 (60 μg / mL), with the lowest viral load in the sample being approximately 0.21 × 10⁻⁶ copies / mL. 4 copies / mL.

[0066] 4. Detection of Astragalus and Curcuma zedoaria extracts on the mRNA levels of PRRSV-infected PAMs cytokines

[0067] Following the optimal mechanism and dosage of action of Astragalus membranaceus and Curcuma zedoaria extract (60 μg / mL) against PRRSV, Pams-163 cells were evenly seeded in 12-well plates and divided into 5 groups. One group was inoculated with PRRSV virus solution, and the other 4 groups were inoculated with different ratios of Astragalus membranaceus and Curcuma zedoaria extract (Curcuma zedoaria: Astragalus membranaceus = 1:2-5) + PRRSV virus solution, with 3 replicates per group. After adding the virus solution (MOI = 0.1) and incubating at 37°C for 1.5 h, the cells were washed 3 times with PBS, and then the Astragalus membranaceus and Curcuma zedoaria extract was added. After incubation at 37°C for 24 h, Pams-163 cells were lysed with Trizol reagent and RNA was extracted. Subsequently, the Ct values ​​of IL-1β, TNF-α, and IFN-α in the cells were detected by RT-qPCR. In addition, the Ct value of porcine β-actin was detected. －ΔΔCt Calculate the relative change. Primers designed for RT-qPCR are shown in Table 2.

[0068] Table 2 RT-qPCR Primers

[0069]

[0070] The results are as follows Figure 4 As shown, compared with the virus control group, the levels of TNF-α mRNA in cells significantly decreased after treatment with the virus in all ratios of Curcuma zedoaria and Astragalus membranaceus extracts, with the most significant decrease observed at Curcuma zedoaria:Astragalus membranaceus ratios of 1:4 and 1:5. The levels of IL-1β mRNA in cells also significantly decreased after treatment with the virus in all ratios of Curcuma zedoaria and Astragalus membranaceus extracts (p < 0.01), with the most significant decrease observed at ratios of 1:4 and 1:5. When the ratio of Curcuma zedoaria and Astragalus membranaceus in the extracts was 1:4 and 1:5, the levels of IFN-α mRNA were significantly upregulated, with a more significant increase observed at a ratio of 1:4 (p < 0.001).

[0071] In summary, in vitro experiments on the inhibition of PRRSV by cytokine assays showed that when the ratio of Curcuma zedoaria to Astragalus membranaceus in the extract was 1:4 and 1:5, it maximally inhibited the pro-inflammatory factors IL-1β and TNF-α, while the ratio of Curcuma zedoaria to Astragalus membranaceus at 1:4 significantly upregulated IFN-α levels. Therefore, the optimal ratio of Curcuma zedoaria to Astragalus membranaceus extract selected in in vitro experiments was 1:4.

[0072] Experimental Example 3: In vitro controlled trial of the effects of Astragalus membranaceus extract and Curcuma zedoaria extract alone.

[0073] To investigate the difference in efficacy between the combined use of Astragalus and Curcuma zedoaria extracts and their individual use, this study conducted a control experiment simultaneously using Astragalus extract and Curcuma zedoaria extract on a Pams-163 microarray. Astragalus extract and Curcuma zedoaria extract were prepared using the decoction method described in Experimental Example 2, and all extracts were stored at room temperature. The concentrations of Astragalus extract and Curcuma zedoaria extract used were 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 60 μg / mL, 30 μg / mL, 15 μg / mL, and 7.5 μg / mL.

[0074] 1. The effects of different concentrations of Astragalus membranaceus extract and Curcuma zedoaria extract on Pams-163 were determined by CCK-8 assay.

[0075] Pams-163 cells were seeded in 96-well plates and, when they reached approximately 90% confluence, the cell culture medium was discarded, and the cells were washed three times with PBS. The extract was serially diluted two-fold with cell maintenance medium, and 100 μL of the extract-containing cell maintenance medium was added to each well of a 96-well plate containing a Pams-163 monolayer. Six replicates were performed for each concentration. A cell control (cells + culture medium + CCK8 solution) and a control without extract (culture medium + CCK8) were also included. After incubation at 37°C for 48 h, CCK-8 reagent (10 μL / well) was added. After incubation at 37°C for 1 h, the OD value was measured at 450 nm using a microplate reader. The cell viability after treatment with different concentrations of extract was calculated.

[0076] Cell viability (%) = (OD value of experimental group - OD value of blank group well) / (OD value of control well - OD value of blank well) × 100%.

[0077] The results are as follows Figure 5 As shown, when the concentration of Astragalus membranaceus extract is ≤60μg / mL, the survival rate of Pams-163 is ≥0.9, so the maximum safe concentration of Astragalus membranaceus extract for Pams-163 is 60μg / mL; when the concentration of Curcuma zedoaria extract is ≤30μg / mL, the survival rate of Pams-163 is ≥0.9, so the maximum safe concentration of Curcuma zedoaria extract for Pams-163 is 30μg / mL.

[0078] 2. Screening of the mechanisms of action of Astragalus membranaceus extract and Curcuma zedoaria extract against PRRSV

[0079] Pams-163 cells were seeded in 6-well plates and cultured until 80% confluence. The following groups were then established: a virus infection group (PRRSV treated alone), a direct drug action group (extract and virus mixed in equal proportions), a drug prevention group (extract first, then virus), a drug inhibition group (virus first, then extract), a drug adsorption group (virus and extract mixed and adsorbed at 4°C), and a blank control group. The treatment methods for Astragalus membranaceus extract and Curcuma zedoaria extract were the same as in Example 2. Specifically, the concentration gradients for Astragalus membranaceus extract were 60 μg / mL, 30 μg / mL, 15 μg / mL, and 7.5 μg / mL; the concentration gradients for Curcuma zedoaria extract were 30 μg / mL, 15 μg / mL, and 7.5 μg / mL.

[0080] The results are as follows Figure 6 Neither Astragalus membranaceus extract nor Curcuma zedoaria extract could inhibit PRRSV viral copy number on Pams-163 (P > 0.05). The concentrations of Astragalus membranaceus extract and Curcuma zedoaria extract, at 60 μg / mL and 30 μg / mL respectively, maximally reduced PRRSV copy number. Therefore, both Astragalus membranaceus extract and Curcuma zedoaria extract have significant antiviral effects in vitro, with inhibition being the optimal mode of action, although their effects are not as pronounced as those of Astragalus membranaceus extract alone.

[0081] 3. Detection of Astragalus membranaceus extract and Curcuma zedoaria extract on the mRNA of PRRSV-infected Pams cytokines

[0082] Based on the optimal mechanism of action and dosage of the two selected extracts against PRRSV, Pams-163 cells were evenly seeded in 12-well plates and divided into three groups: Group 1 was inoculated with PRRSV virus solution; Group 2 was inoculated with Astragalus membranaceus extract (60 μg / mL) + PRRSV virus solution; and Group 3 was inoculated with Curcuma zedoaria extract (30 μg / mL) + PRRSV virus solution, with three replicates per group. After incubation at 37°C for 24 h, Pams-163 cells were lysed with Trizol reagent to extract RNA, which was then analyzed by 2... －ΔΔCt Changes in cytokine mRNA levels were calculated; all primers are shown in Table 2.

[0083] The results are as follows Figure 7 As shown, compared with the virus control group, the levels of IL-1β and TNF-α mRNA in cells were significantly decreased after Astragalus extract and Curcuma zedoaria extract inhibited PRRSV, but the degree of decrease was far less than that of Astragalus extract and Curcuma zedoaria extract. In addition, Astragalus extract alone could significantly upregulate IFN-α mRNA levels, but the degree of upregulation was far less than that of Astragalus extract and Curcuma zedoaria extract, while Curcuma zedoaria extract alone did not show a significant difference compared with the virus group.

[0084] In summary, comparing the in vitro antiviral and immunomodulatory effects of Astragalus and Curcuma zedoaria extracts, both combined and individual, revealed that when PRRSV virus fluid was inoculated at the same MOI, both extracts significantly inhibited PRRSV replication in vitro. However, the combined effect resulted in the lowest PRRSV copy number, ranging from 0.21 to 1.37 × 10⁻⁶. 4 The lowest PRRSV copy numbers were 4.27–7.41 × 10⁻⁶ copies / mL, while the lowest copy numbers of PRRSV when the two extracts were used alone were 4.27–7.41 × 10⁻⁶ copies / mL. 4 copies / mL (Astragalus extract), 2.57–4.27 × 10 5 The concentration of copies / mL (Curcuma zedoaria extract) indicates that the combined effect of the two extracts significantly enhances the virus-inhibiting ability, which is superior to the effect of either extract alone. Furthermore, both the combined effect and the individual effects of the two extracts significantly downregulated IL-β and TNF-α mRNA levels in vitro, but the combined effect of Curcuma zedoaria and Astragalus membranaceus extracts at ratios of 1:4 and 1:5 significantly upregulated IFN-α mRNA levels, while the IFN-α mRNA level did not change significantly when Curcuma zedoaria extract was used alone.

[0085] Example 4: A Preliminary Study on the Use of Astragalus and Curcuma Extracts as Enhancers for Piglet Vaccines

[0086] 1. Animal experiments

[0087] Using PRRSV inactivated vaccine (classic CH-1a strain) and the optimal ratio of Astragalus membranaceus and Curcuma zedoaria screened by cell experiments, an animal experiment was designed as follows:

[0088] Table 3 Grouping of Animal Experiments

[0089]

[0090] Six 4-week-old piglets were randomly assigned to each group. All piglets were fed and had access to water normally. Piglets in groups B and C were vaccinated at 4 weeks of age, with the dosage and method of vaccination following the manufacturer's instructions. Group A received the same dose of diluted vaccine.

[0091] Group C piglets were fed with Astragalus and Curcuma extracts. According to Part II of the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China, the intake of Astragalus slices per pig should not exceed 15g / day, and the intake of Curcuma slices per pig should not exceed 10g / day. The daily intake of Astragalus and Curcuma extracts was calculated based on the concentration of the extracts.

[0092] 2. Clinical symptom observation

[0093] Pigs were monitored daily, and their physical condition and clinical scores were recorded based on behavior, respiratory symptoms, and coughing. Rectal temperature and clinical symptoms were observed and recorded daily. All piglets were weighed at the beginning and end of the trial to calculate average daily weight gain. The trial ended 28 days after vaccination.

[0094] After vaccination, clinical symptoms were scored daily using a clinical symptom scoring scale. The results were as follows: Figure 8 A. After vaccination, no clinical symptoms were observed in piglets in groups A, B, and C. However, piglets in groups B and C showed a slight increase in body temperature after vaccination. Figure 8 Group B), whose body temperature persisted until day 4, may be an immune response to the vaccine in piglets. Over time, the body temperatures of piglets in groups B and C converged to similar levels, showing no significant difference. This indicates that, in the long term, vaccination and feeding with Astragalus and Curcuma extract have no effect on the body temperature and clinical symptoms of piglets. All piglets were weighed on the day of vaccination and on day 28, and the average daily weight gain was calculated. The results are as follows: Figure 8 As shown in Figure C, the results indicate that, compared with the control group, the average daily weight gain of piglets in group C (fed with Astragalus and Curcuma extract) was significantly increased (p < 0.05).

[0095] 3. Detection of PRRSV antibody levels and cytokines

[0096] Piglet serum was collected at 0, 7, 14, 21, and 28 days post-vaccination to assess the promoting effect on PRRSV antibody levels and cytokine secretion levels as screening indicators. PRRSV N protein-specific antibodies were detected using the PRRSV universal antibody detection kit (C2) from Shenzhen Kangbaide Biotechnology Co., Ltd. PRRSV antibody results were reported as the sample value / positive value (S / P) ratio; a sample S / P ratio ≥ 0.2 was considered PRRSV antibody positive. Piglet serum collected at each time point was analyzed for IL-1β, IL-6, IL-4, TNF-α, and IL-2 cytokine secretion levels according to the instructions of the respective cytokine kits.

[0097] PRRSV antibody results are as follows Figure 9 As shown, the results indicated that: throughout the experiment, no PRRSV antibodies were detected in group A (blank control group), indicating that there was no interference from live PRRSV virus during the experiment, and the experiment started in a healthy, virus-free state; in group B (vaccine group), the PRRSV antibody level gradually increased over time, with the average S / P ratio of piglet serum antibodies greater than 0.2 on day 14 after vaccination, and all piglets were positive for PRRSV antibodies 21 days after vaccination; in group C (vaccine + Astragalus and Curcuma extract group), the PRRSV antibody level was significantly higher than that in group B (vaccine group) starting from day 7 after vaccination (p≤0.05), and the average S / P ratio was greater than 0.2. The antibody level in group C differed most from that in group B at day 28 (p≤0.0001). In summary, the antibody level in group C 28 days after vaccination was significantly higher than that in group B (p≤0.0001), indicating that Astragalus and Curcuma extract may have a positive effect on improving the immune response of piglets to PRRSV, suggesting that it is a beneficial immune enhancement strategy.

[0098] The results are as follows Figure 10As shown, the IL-1β secretion levels of piglets in each group were analyzed. Compared with group A (blank control group), the IL-1β levels of piglets in other groups generally showed an increasing trend within 14 days. By day 21, group C (vaccine + Astragalus and Curcuma extract group) showed a significant decrease in IL-1β compared with group B (p≤0.05), indicating that Astragalus and Curcuma extract can significantly reduce IL-1β secretion levels 21 days after vaccination. The IL-6 secretion levels of piglets in each group were also analyzed compared with group A (blank control group). In other groups of piglets, IL-6 generally showed an increasing trend over 14 days. From day 21, compared with group B (vaccinated group), the IL-6 secretion level in group C piglets was significantly lower (p≤0.0001), indicating that Astragalus and Curcuma extract can significantly reduce IL-6 secretion levels 21 days after vaccination. Analysis of IL-4 secretion levels in each group of piglets showed that from day 7 after vaccination, compared with group B (vaccinated group), the IL-4 secretion level in group C piglets significantly increased, and this increase continued over time. Over time, IL-4 secretion levels gradually increased, indicating that Astragalus and Curcuma extract significantly enhanced IL-4 secretion levels in piglets after vaccination. Analysis of TNF-α secretion levels in each group showed that, compared to group A (blank control group), TNF-α levels in other groups generally increased over 14 days. From day 7, compared to group B (vaccinated group), TNF-α levels in group C were significantly lower, reaching their lowest point at day 28, with TNF-α secretion levels lower than in group A (blank control group). This indicates that, compared to vaccination alone, Astragalus and Curcuma extract significantly and continuously reduced TNF-α secretion levels in piglets starting 7 days after vaccination. Analysis of IL-2 secretion levels in each group showed that, compared to group A, groups B and C showed significant increases starting 14 days (p≤0.001). After day 21, IL-2 secretion levels in group C were significantly higher than in the vaccinated group, indicating that, compared to vaccination alone, Astragalus and Curcuma extract significantly and continuously increased IL-2 secretion levels in piglets starting 21 days after vaccination.

[0099] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The application of a drug extract in enhancing the efficacy of porcine reproductive and respiratory syndrome (PRRS) vaccine, characterized in that, The drug extract is an aqueous extract of Astragalus membranaceus and Curcuma zedoaria, wherein the mass ratio of Astragalus membranaceus to Curcuma zedoaria is 4-5:1; The method for preparing the drug extract includes: Place Astragalus membranaceus and Curcuma zedoaria in water at a mass ratio of 4-5:1 and simmer over low heat several times. Collect the filtrate, concentrate it, dry the resulting extract, granulate it, and obtain Astragalus membranaceus and Curcuma zedoaria extract.

2. The application according to claim 1, characterized in that, The drug extract can enhance the ability to suppress the virus after the use of a porcine reproductive and respiratory syndrome vaccine.

3. The application according to claim 1, characterized in that, The drug extract significantly downregulated the mRNA levels of IL-β and TNF-α and upregulated the mRNA level of IFN-α after administration of the porcine reproductive and respiratory syndrome vaccine.

4. The application according to claim 1, characterized in that, The concentration of the drug extract is 50~60 μg / mL.

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