Application of polysaccharide from panax japonicus in preparation of medicine for resisting porcine reproductive and respiratory syndrome virus

Abstract

This invention discloses the application of *Gynostemma pentaphyllum* polysaccharide in the preparation of drugs against porcine reproductive and respiratory syndrome virus (PRRSV). This invention explores the regulatory effect of *Gynostemma pentaphyllum* polysaccharide on PRRSV replication, and for the first time discovers that *Gynostemma pentaphyllum* polysaccharide can effectively inhibit PRRSV replication in Marc-145 cells. Based on this, this invention proposes a new application of *Gynostemma pentaphyllum* polysaccharide in the preparation of drugs against PRRSV, preferably with a concentration of 12.5 μg / mL to 100 μg / mL. This invention enriches the research on the antiviral activity of *Gynostemma pentaphyllum* polysaccharide and also provides a new technical means for the research and clinical application of anti-PRRSV drugs.

Description

Technical Field

[0001] This invention relates to a novel anti-porcine reproductive and respiratory syndrome virus (PRRSV) drug, and particularly to the application of *Panax japonicus* polysaccharide in the preparation of such an anti-PRRSV drug. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). It is distributed worldwide and has caused enormous losses to the global pig industry. The main symptoms include reproductive disorders in sows, such as abortion, stillbirth, and mummified fetuses, and severe respiratory diseases and slow growth in piglets. PRRSV belongs to the order Nidovirales, family Arteriviridae, and genus Arterivirus; it is a single-stranded positive-sense RNA virus. PRRSV is characterized by rapid mutation, multiple lineages, and a high likelihood of producing recombinant strains. Furthermore, the cross-protective properties between different lineages are weak, making vaccine-based prevention and control increasingly difficult. Developing safe and effective anti-PRRSV drugs is of great significance for the prevention and control of PRRSV.

[0003] Traditional Chinese medicine (TCM) has many advantages in antiviral treatment, including high efficacy, low toxicity, and low cost. Panax japonicus (T.Nees) CAMey., also known as "Bamboo Joint Ginseng," "Seven-Leaf," and "White Sanqi," belongs to the genus Panax in the family Araliaceae. It was first recorded in Zhao Xuekai's *Bai Cao Jing* (Mirror of Herbs). Panax japonicus has a sweet and slightly bitter taste, is warm in nature, and possesses a wide range of pharmacological activities, including preventing ulcers, protecting the liver, promoting gastrointestinal motility, sedation and sleep induction, anticonvulsant effects, fatigue relief, lowering blood lipids, protecting the cardiovascular system, enhancing immunity, analgesia, anti-inflammation, anti-tumor effects, and delaying aging. Panax japonicus is rich in diverse components, including saponins, sugars, amino acids, volatile oils, nucleosides, and inorganic salts. Phytochemical studies have shown that the main active components of Panax japonicus are saponins from Panax japonicus and polysaccharides from Panax japonicus (PSPJ). The total saponin content in the root of *Panax notoginseng* is as high as 15%, which is 2 to 7 times that of ordinary ginseng. The medicinal effects of *Panax notoginseng* mainly come from its tetracyclic or pentacyclic triterpenoid saponins, while polysaccharides are complex macromolecular sugars with the general formula (C6H2O). 12 O6) x . Summary of the Invention

[0004] The purpose of this invention is to provide a novel use of ginseng polysaccharide (PSPJ) in the preparation of drugs against porcine reproductive and respiratory syndrome virus.

[0005] To achieve the above objectives, the present invention employs the following technical means.

[0006] This invention investigates the regulatory effect of different concentrations of PSPJ (12.5 μg / mL to 100 μg / mL) on PRRSV replication. The results showed that all concentrations of PSPJ within the measured range inhibited PRRSV replication, and the inhibitory effect gradually increased with increasing PSPJ concentration. This indicates that PSPJ can effectively inhibit PRRSV proliferation in a concentration-dependent manner, with 100 μg / mL PSPJ showing the best antiviral effect. Marc-145 cells inoculated with LN1701 were treated with higher concentrations of PSPJ (50 μg / mL and 100 μg / mL). After 48 h of culture, the effect of the drug on PRRSV N protein expression was detected by IFA assay. The results showed that compared with the control group, N protein expression gradually decreased with increasing PSPJ concentration, and this decreasing trend was positively correlated with PSPJ concentration. This indicates that PSPJ can inhibit PRRSV proliferation in Marc-145 cells in a concentration-dependent manner. Further investigation was conducted into the effect of PSPJ on the PRRSV proliferation curve. Marc-145 cells were seeded with LN1701 and then treated with 100 μg / mL PSPJ. Cells were harvested at 12 h, 24 h, 36 h, and 48 h post-infection for TCID assay. 50 Experiments showed that PSPJ treatment significantly inhibited viral replication at 24h, 36h, and 48h post-infection, with viral titers decreasing 10-fold at 24h, 8.91-fold at 36h, and 3.76-fold at 48h.

[0007] Based on the above research, this invention proposes the application of *Panax japonicus* polysaccharide in the preparation of drugs against porcine reproductive and respiratory syndrome virus.

[0008] Preferably, the *Panax japonicus* polysaccharide can significantly inhibit PRRSV replication in Marc-145 cells.

[0009] Preferably, the concentration of the *Panax japonicus* polysaccharide is 12.5 μg / mL to 100 μg / mL.

[0010] Compared with the prior art, the beneficial effects of the present invention are:

[0011] This invention explores the regulatory effect of *Gynostemma pentaphyllum* polysaccharide on PRRSV replication, and for the first time discovers that *Gynostemma pentaphyllum* polysaccharide can effectively inhibit PRRSV replication in Marc-145 cells. Based on this, this invention proposes a new application of *Gynostemma pentaphyllum* polysaccharide in the preparation of drugs against porcine reproductive and respiratory syndrome virus (PRRSV). This invention enriches research on the antiviral activity of *Gynostemma pentaphyllum* polysaccharide and also provides new technical means for the research and clinical application of anti-PRRSV drugs. Attached Figure Description

[0012] Figure 1 Comparison of viability of Marc-145 cells with different concentrations of PSPJ;

[0013] Figure 2 To investigate the effect of different doses of PSPJ on PRRSV replication (ORF7 mRNA level);

[0014] Figure 3 To investigate the effect of different doses of PSPJ on PRRSV replication (N protein expression level);

[0015] Among them, A: IFA identification of the effect of different concentrations of PSPJ treatment on PRRSV N protein expression; B: Statistical results of fluorescence counts;

[0016] Figure 4 To investigate the effect of PSPJ on the PRRSV proliferation curve. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0018] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0019] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0020] Example 1: Application of Papaya Polysaccharide (PSPJ) in the treatment of porcine reproductive and respiratory syndrome virus (PRRSV)

[0021] 1. Materials

[0022] 1.1 Source of cells and viruses used in the experiment

[0023] Marc-145 cells preserved in the laboratory were cultured in DMEM medium (10% FBS); the PRRSV-LN1701 strain was isolated in our laboratory.

[0024] 1.2 Main Instruments

[0025] Table 1 Main Instruments

[0026]

[0027]

[0028] 1.3 Main reagents and their preparation

[0029] 1.3.1 Main Reagents

[0030] Table 2 Main Reagents

[0031]

[0032] 1.3.2 Reagent Preparation

[0033] (1) 10% DMEM cell culture medium: Add 10% fetal bovine serum to DMEM cell culture medium, mix well, and store at 4℃ for later use.

[0034] (2) 5 mg / mL MTT colorimetric solution: Dissolve 0.5 g MTT in a warm water bath under light-protected conditions, make up to 100 mL of PBS buffer, filter to sterilize, and store in the dark.

[0035] (3) PBS Phosphate Buffer: Weigh 7.9g NaCl, 0.2g KCl, 0.24g KH2PO4 (or 1.44g Na2HPO4), and 1.8g K2HPO4, dissolve them in 800mL distilled water, adjust the pH of the solution to 7.4, and add distilled water to a final volume of 1L. After volume adjustment, dispense and autoclave. The sterilized solution should be stored at 4℃, and its maximum shelf life is one month.

[0036] 2 methods

[0037] 2.1 Culture of Marc-145 cells

[0038] 2.1.1 Resuscitation of Marc-145 cells

[0039] (1) Marc-145 cells were removed from liquid nitrogen and thawed quickly in a constant temperature water bath at 37°C.

[0040] (2) After it is completely thawed, place it in a low-speed centrifuge and centrifuge at 1000 rpm for 5 min, then discard the supernatant.

[0041] (3) After resuspending the cells in about 1 mL of 10% cell culture medium, transfer them to a 25T cell culture flask and bring the culture medium to 5 mL. Place the cells in an incubator and culture them at 37°C and 5% CO2.

[0042] 2.1.2 Passaging of Marc-145 cells

[0043] (1) Observe the cell growth status every day. Once the Marc-145 cells cultured in the cell flask have grown to a monolayer, they can be passaged.

[0044] (2) Use a pipette to remove and discard the culture medium in the bottle. Add 1 mL of trypsin to the 25T cell bottle and digest for 1-3 min. When the cells are spherical and the gaps between the cells are significantly widened, add 1 mL of growth medium to stop the digestion process.

[0045] (3) After digestion is terminated, use a pipette to thoroughly blow the cells until they are completely detached. Then, add an appropriate amount of 10% cell culture medium to resuspend the cells and continue culturing.

[0046] 2.1.3 Cryopreservation of Marc-145 cells

[0047] Marc-145 cells cultured to a monolayer were first digested with trypsin. After digestion, growth medium was added to stop the digestion. The solution containing cells was transferred to a low-speed centrifuge and centrifuged at 1000 rpm for 5 minutes, discarding the supernatant. After adding cryopreservation solution, the cells were aliquoted and carefully labeled. The cryovials were placed in a -80°C freezer overnight, and then transferred to liquid nitrogen for long-term storage the next day.

[0048] 2.2 Cytotoxicity assay of Panax notoginseng polysaccharide (PSPJ)

[0049] 2.2.1 Dissolution of PSPJ (Polygonum multiflorum polysaccharide)

[0050] PSPJ was dissolved in DMSO to achieve an initial concentration of 100 mg / mL, followed by serial dilutions to determine the drug's cytotoxicity.

[0051] 2.2.2 Cytotoxicity assay of Panax notoginseng polysaccharide

[0052] (1) Cell seeding and treatment: Marc-145 cells were evenly seeded on 96-well plates. After the cells adhered, PSPJ solution (100 μL / well) at six different concentration gradients was added. The control group (Mock) was given an equal amount of cell maintenance medium (100 μL / well). Four replicates were set for each concentration.

[0053] (2) Culture and observation: The culture plate was placed in a 37℃, 5% CO2 incubator for 72h, and the cell status was observed daily (e.g., whether it detached or disintegrated).

[0054] (3) MTT assay: Discard the culture medium, add 25 μL of MTT solution to each well, and continue culturing for 4 h. Discard the MTT solution, add 150 μL of LDMSO to dissolve the formazan crystals, and let stand at 37 °C for 30 min.

[0055] (4) Data analysis: The absorbance (OD value) at a wavelength of 490 nm was measured using an ELISA reader. Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%. Maximum safe concentration (MNTC): The highest drug concentration at which cell viability ≥ 90% is achieved.

[0056] 2.3 Determination of the effect of different doses of Panax notoginseng polysaccharide on PRRSV

[0057] (1) Indirect immunofluorescence (IFA)

[0058] a. Virus inoculation and infection

[0059] The supernatant (1 MOI) of the 5th generation LN1701 virus strain was inoculated into a 24-well plate containing Marc-145 cells and incubated for 2 hours, with gentle shaking every 15 minutes. The supernatant was discarded, and the cells were washed twice with PBS and then replaced with maintenance medium. The cells were cultured for another 48 hours until cytopathic effect (CPE) was evident.

[0060] b. Cell fixation

[0061] Discard the culture medium and wash the cells three times with pre-cooled PBS. Add 200 μL of cell fixative to each well and fix for 10 min. Discard the fixative and wash three more times with PBS (5 min each time). Add 0.1% Triton X-100 (400 μL / well) and incubate at room temperature for 10 min (shake).

[0062] c. Blocking and antibody incubation

[0063] Block with 2% BSA for 30 min (room temperature), wash 3 times with PBS. Add PRRSVN protein monoclonal antibody (1:1000), incubate at 4°C for 12 h, wash 3 times with PBS. Add FITC-labeled secondary antibody (1:200, 150 μL / well), incubate in the dark for 1 h, wash 3 times with PBS.

[0064] d. Observation of results

[0065] The culture plate was placed under an inverted fluorescence microscope to observe the fluorescence and the virus titer was calculated.

[0066] (2) Quantitative Real-Time PCR (RT-qPCR)

[0067] After freezing and thawing the sample three times, centrifuge and collect the supernatant for RNA extraction using the Trizol method. RT-qPCR was then performed. The components were added as required (Table 4), mixed thoroughly, briefly centrifuged, and then placed in a quantitative PCR instrument. The program was set according to Table 5. After the experiment, the CT values ​​were exported and analyzed using 2... －△△CT The method is to conduct data analysis.

[0068] Result determination: If the intrinsic parameter values ​​are uniform, the test sample curve is a standard S-shaped curve, and the CT value of the test result is ≤38, then the result is reliable.

[0069] Table 3 Primers required for RT-qPCR reaction

[0070]

[0071] Table 4 RT-qPCR reaction system

[0072]

[0073] Table 5. Reaction conditions for quantitative real-time PCR

[0074]

[0075] (3)TCID 50 test

[0076] a. Virus dilution and inoculation

[0077] When the cell density in the 96-well plate reaches 70-80%, the virus solution frozen at -80℃ is thawed in an ice bath. The solution is then serially diluted 10-fold to seven concentrations (100 μL virus solution + 900 μL maintenance solution). Each dilution is replicated in eight wells, and each well is inoculated with 100 μL of diluted virus solution. A negative control well is also included.

[0078] b. Cultivation and Fixation

[0079] Incubate at 37℃ and 5% CO2 for 48 h, discard the culture medium, wash three times with PBS, and add 50 μL of fixative to each well for 10 min.

[0080] c. Immunofluorescence staining

[0081] After washing with PBS, the sample was treated with 0.1% Triton X-100 for 10 min, blocked with 2% BSA for 30 min, incubated with primary antibody (1:1000) at 4℃ for 12 h, and then incubated with FITC-labeled secondary antibody (1:200) in the dark for 1 h.

[0082] d. Result determination

[0083] The culture plate was placed under an inverted fluorescence microscope to observe the fluorescence and the virus titer was calculated.

[0084] 3 Results and Analysis

[0085] 3.1 Maximum safe concentration of *Gynostemma pentaphyllum* polysaccharide in Marc-145 cells

[0086] To investigate the effect of different concentrations of PSPJ on the viability of Marc-145 cells, PSPJ was diluted to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. Cell viability was assessed using the MTT assay to determine the maximum safe concentration of PSPJ in Marc-145 cells. Results are as follows: Figure 1 As shown in the table, there was no difference in cell viability between Marc-145 cells treated with PSPJ at concentrations of 100 μg / mL and the control group. However, when the PSPJ concentration was increased to 200 μg / mL, cell viability was below 90%, indicating that PSPJ at concentrations of 100 μg / mL and below had no significant effect on the viability of Marc-145 cells.

[0087] 3.2 Effects of different doses of Panax notoginseng polysaccharide on PRRSV replication

[0088] To assess the effect of PSPJ on PRRSV replication, this study used RT-qPCR, IFA, and TCID assays, respectively. 50 The effects of PSPJ on PRRSV ORF7 gene levels, N protein expression levels, and viral titers were analyzed as follows:

[0089] Marc-145 cells were seeded with LN1701 cells and then treated with PSPJ at concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. After 48 h, cell samples were collected, and the PRRSV ORF7 gene level in each group was detected using RT-qPCR. Figure 2 The results showed that different concentrations of PSPJ could inhibit the PRRSV ORF7 gene level, and the inhibitory effect on the virus gradually increased with increasing PSPJ concentration. This indicates that PSPJ can effectively inhibit PRRSV proliferation, and the inhibitory effect is concentration-dependent, with 100 μg / mL PSPJ showing the best inhibitory effect.

[0090] Marc-145 cells seeded with LN1701 were treated with high concentrations of PSPJ (50 μg / mL and 100 μg / mL). After 48 h of culture, the effect of the drug on PRRSVN protein expression was detected by IFA assay. Results are as follows: Figure 3As shown, compared with the control group, the expression level of N protein gradually decreased with increasing PSPJ concentration, and this decreasing trend was positively correlated with the PSPJ concentration. This further confirms that PSPJ can inhibit the proliferation of PRRSV in Marc-145 cells, and this inhibitory effect is concentration-dependent.

[0091] Marc-145 cells were inoculated with LN1701 and then treated with 100 μg / mL PSPJ. Cells were harvested at 12 h, 24 h, 36 h, and 48 h post-infection to detect the effect of PSPJ on viral proliferation curves. Results are as follows: Figure 4 As shown, the PSPJ treatment group significantly inhibited viral replication at 24h, 36h and 48h post-infection, with viral titers decreasing 10-fold at 24h, 8.91-fold at 36h and 3.76-fold at 48h.

[0092] 4. Summary

[0093] 4.1 The maximum safe concentration of PSPJ in Marc-145 cells is 100 μg / mL.

[0094] 4.2 PSPJ can significantly inhibit the replication of PRRSV in Marc-145 cells, and its inhibitory effect shows a clear concentration gradient dependence (12.5 μg / mL to 100 μg / mL).

Claims

1. Application of Panax notoginseng polysaccharide in the preparation of drugs against porcine reproductive and respiratory syndrome virus (PRRSV).

2. Use according to claim 1, wherein The concentration of the *Panax japonicus* polysaccharide is 12.5 μg / mL to 100 μg / mL.

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