Application of Saxifraga herba water extract in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs

Abstract

The invention discloses an application of a Saxifrage water extract in the preparation of an anti-porcine reproductive and respiratory syndrome virus drug, and relates to the technical field of antiviral treatment. The invention applies the Saxifrage water extract to the anti-porcine reproductive and respiratory syndrome virus drug, which has little cytotoxic side effects and has good anti-PRRSV activity, thus opening up new application prospects for PRRS prevention and control.

Description

Technical Field

[0001] The invention relates to the technical field of antiviral treatment, and in particular to application of a saxifrage water extract in the preparation of an anti-porcine reproductive and respiratory syndrome virus drug. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease that causes abortion in sows, high fever, interstitial pneumonia and growth retardation in weaned piglets. Since PRRS-infected pigs show typical clinical symptoms of cyanosis of the ears, the disease is commonly known as "blue ear disease". At present, my country's PRRS prevention and control strategy is still mainly based on vaccine immunization. To date, there are two types of commercial PRRS vaccines used in clinical practice in my country: inactivated vaccine (KV) and attenuated live vaccine (MLV). KV vaccines are made by inactivating the parent strains of C-PRRSV CH-1a and HP-PRRSVJXA1 in my country. MLV vaccines are attenuated by continuous cell passage, and there are seven main types: Ingelvac PRRSVMLV (parent strain VR-2332), CH-1R (CH-1a), R98 (R98), and GDr180 (GD). Since 2011, several attenuated live vaccines (MLVs) based on HP-PRRSV as parent strains have been widely used in clinical practice as commercial vaccines in my country. These MLVs can provide good immune protection against homologous strains in clinical practice, reduce the outbreak of HP-PRRS, and play a huge role in preventing and controlling the occurrence of the disease in my country. However, with the large-scale, long-term, and irrational use of MLV vaccines in my country, coupled with the characteristics of easy mutation and antibody-dependent enhancement (ADE) of PRRSV strains, not only has the immune selection pressure in the pig population increased, but also the virus has begun to undergo genetic mutation and evolution. At present, there are four main problems with MLV vaccines in clinical practice: (1) poor cross-protection. MLV can provide good protection against homologous strains in clinical practice, but cannot provide good cross-protection against heterologous strains; (2) reverse virulence. Long-term use of MLV or use in PRRS-negative fields may lead to reverse virulence, which in turn causes PRRS outbreaks; (3) genetic recombination. Clinical strain isolation monitoring shows that a large number of recombinant strains between wild strains and MLV have appeared in clinical practice in my country, especially the emergence of NADC30-like strains and QYYZ-like strains; (4) Vertical transmission. Sows vaccinated with MLV vaccine can transmit the virus to newborn piglets, which may cause clinical symptoms of PRRS in piglets.

[0003] In recent years, in addition to the development of viral vaccines, the research on antiviral drugs has also developed by leaps and bounds, playing a huge role in the prevention and treatment of viral diseases in clinical practice. Traditional Chinese medicines (TCM) have a long history of use in my country and are rich in resources. These drugs have long-term clinical trials and reliable efficacy, so they are considered to be the source of new antiviral drugs. As early as 1969, the team of Chinese scientific researcher Tu Youyou began to try to extract antimalarial drugs from traditional Chinese medicine. Until 2015, Tu Youyou was awarded the Nobel Prize for extracting artemisinin (C 15 H 22 O5) won the Nobel Prize for its important role in anti-malarial treatment, and the research and development of Chinese medicine in my country has once again attracted widespread attention. At present, the research on TCM is increasing at home and abroad. Many studies have found that TCM has a broad-spectrum antiviral effect. Screening out effective antiviral ingredients from it has become a new direction for the development of antiviral drugs. However, there are currently more studies on anti-human virus drugs, while the research and development of anti-animal virus drugs is relatively small.

[0004] PRRS is an immunosuppressive disease of pigs, which has caused great economic losses to the world's pig industry. At present, there are more and more studies on anti-PRRSV chemical synthetics, PRRSV cell entry blockers and nanobodies, but these studies are still in the laboratory research or clinical trial stage. Due to the limited protection of the current commercial MLV vaccine and the frequent emergence of PRRSV mutant strains, MLV vaccine alone cannot provide completely effective protection. Therefore, the development of effective, economical and practical anti-PRRSV drugs is of great significance for the prevention and control of PRRS in my country. At present, herbal medicine based on traditional Chinese medicine has been widely used as a source of new drug research and development, and many Chinese herbal medicine extracts have been shown to inhibit PRRSV infection, such as honeysuckle, scutellaria, and lianchi, which have the effect of clearing heat and detoxifying, and cinnamon twig, ephedra, and bupleurum, which have the effect of dispelling exterior pathogens. In 2013, Cheng et al. isolated 17 compounds from traditional Chinese medicine that were proven to have multiple pharmacological activities, such as antiviral, anti-inflammatory and immunomodulatory. Their anti-PRRSV activity was evaluated in vitro based on Marc-145 cells. The results showed that chlorogenic acid and scutellarin had potential anti-PRRSV activity, and their antiviral mechanism may be that the drugs directly inactivate or interfere with the early replication stage of the virus. Tanshinone IIA is one of the most abundant components in the root of Salvia miltiorrhiza. Sun et al. (2014) confirmed that Tanshinone IIA can inhibit PRRSV by inhibiting viral N gene expression and blocking virus-induced cell apoptosis. Epigallocatechin gallate (EGCG), which is rich in tea, has also been shown to inhibit PRRSV replication in MARC-145 cells. Matrine (C15 H 24 N2O) is a drug component isolated and purified from Sophora flavescens. Studies have shown that matrine not only has a significant inhibitory effect on hepatitis B virus (HBV) and hepatitis C virus (HCV), but also has a pharmacological effect of enhancing the body's immune regulation. In 2016, Sun extracted matrine from the root of Sophora flavescens. In vitro studies of PAM cells found that matrine not only has in vitro anti-PRRSV activity, but also has an inhibitory effect on PCV2 infection. Its antiviral mechanism is related to the inhibition of TLR3, 4 / NF-4 / NF-κB / TNF-α pathway expression. Studies have also found that the anti-PRRSV activity of these drugs is non-specific and can simultaneously inhibit the replication of different viruses or different strains of the same virus. In addition, these active drug ingredients not only have antiviral effects, but also have the effect of enhancing the body's immune capacity. Therefore, further research and development of anti-PRRSV drugs will open up new application prospects for PRRS prevention and control. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an application of a water extract of Saxifrage in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs, which has little cytotoxic side effects and has good anti-PRRSV activity, thereby opening up new application prospects for PRRS prevention and control.

[0006] The technical solution of the present invention to solve the above technical problem is as follows: provide an application of a water extract of Saxifraga herba in the preparation of an anti-porcine reproductive and respiratory syndrome virus drug.

[0007] Further, the water extract of Saxifraga herba is prepared by the following method:

[0008] The saxifrage powder was wrapped with filter paper and put into a Soxhlet extractor, and neutral deionized water with a mass of 10 times that of the saxifrage powder was added for extraction for 4-6 hours. After the water extract was recovered, it was concentrated and fixed to volume in a 37° C. water bath to obtain the saxifrage water extract.

[0009] Furthermore, after the volume was fixed, the concentration of Saxifraga was 4-6 g / mL in the concentrated solution.

[0010] Furthermore, after the volume was fixed, the concentration of Saxifraga was 5 g / mL in the concentrated solution.

[0011] An anti-porcine reproductive and respiratory syndrome virus drug comprises the above-mentioned saxifrage water extract.

[0012] The present invention has the following beneficial effects:

[0013] 1. The present invention applies the water extract of Saxifraga to the preparation of anti-porcine reproductive and respiratory syndrome virus drugs, which has little cytotoxic side effects and has good anti-PRRSV activity, opening up new application prospects for PRRS prevention and control.

[0014] 2. An anti-PRRSV drug evaluation system was established using primary PAM cells, and the water extract of Saxifraga heracleifolia was used to evaluate its anti-PRRSV activity. The results showed that the drug extract had little cytotoxicity and side effects and had good anti-PRRSV activity, providing a reference for the development of anti-PRRSV drugs in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the anti-PRRSV activity test of various Chinese herbal medicine extracts;

[0016] Figure 2 The results of the IFA test of Saxifraga hydrophila extract (HEC) against PRRSV;

[0017] Figure 3 To evaluate the activity of Saxifraga herbacea extract (HEC) against different strains of PRRSV. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0019] Example 1

[0020] Application of water extract of Saxifraga herba in the preparation of drugs against porcine reproductive and respiratory syndrome virus.

[0021] Specifically, the water extract of Saxifraga herba is prepared by the following method:

[0022] 100 g of Saxifrage powder was wrapped with filter paper and placed in a Soxhlet extractor. Neutral deionized water (100 mL) with a mass ratio of 10 times that of the Saxifrage powder was added and extracted for 5 h. After the water extract was recovered, it was concentrated and fixed to volume in a 37°C water bath. After fixing to volume, the concentration of Saxifrage in the concentrate was 5 g / mL, and the Saxifrage water extract was obtained.

[0023] Meanwhile, the ethanol extract of Saxifraga sibiricum is prepared by the following method:

[0024] 100 g of Saxifrage powder was wrapped with filter paper and placed in a Soxhlet extractor. 4000 mL of anhydrous ethanol (solid-liquid ratio of 1:40) was added for Soxhlet extraction for 6 h. After recovering the alcohol extract, it was concentrated and fixed to volume in a 37°C water bath. After fixing to volume, the concentration of Saxifrage in the concentrate was 5 g / mL, and the Saxifrage water extract was obtained.

[0025] The method is used to prepare alcohol-extracted crab shell, water-extracted radix scutellariae, water-extracted radix scutellariae, water-extracted saxifrage water extract, water-extracted senecio, water-extracted dry pine, water-extracted alum leaf, water-extracted madder, alcohol-extracted emblica fruit and water-extracted meconopsis pilosa.

[0026] Establishment of antiviral drug screening system

[0027] 1. MTT method to determine drug cytotoxicity

[0028] (1) The primary PAM frozen in liquid nitrogen was rapidly thawed in a 37°C water bath and added to RPMI-1640 medium containing 10% FBS.

[0029] (2) Approximately 1×105 cells were added to each well of a 96-well plate and cultured in a 37°C incubator for 2-3 h. Then, 200 mL / well of fresh culture medium was replaced and the plate was incubated at 37°C for another 24 h.

[0030] (3) The drug (water extract or alcohol extract) was diluted 2-fold in RPMI-1640 medium containing 2% FBS (12 dilution gradients for natural drugs), and 200 μL was added to each well of a 96-well cell culture plate. Four replicates were set for each dilution, and four replicate wells were set for a cell control group.

[0031] (4) After incubation at 37°C for 48 h, the culture medium was discarded and 20 μL of MTT solution was added to each well. After incubation at 37°C for 4 h, the supernatant was removed and 110 μL of Formazan solution was added to each well. The cells were gently shaken for 10 min to completely dissolve the crystals, and the absorbance (OD) was read at 490 nm using an enzyme-linked immunosorbent assay. 490nm ). The higher the absorbance, the less cytotoxic the drug is, while the lower the absorbance, the more cytotoxic the drug is.

[0032] (5) Set up zeroing wells (containing RPMI-1640 culture medium, MTT and Formazan dissolution solution).

[0033] (6) Calculate the cytopathic rate and cell viability rate. Cell viability rate = OD of the experimental group 490nm Value / cell control group OD 490nm The value is 100%. Cytopathic rate = (cell control group OD 490nm Value - test group OD 490nm value) / cell control group OD 490nm The lowest drug dilution concentration with a cell viability rate of more than 90% is taken as the highest safe drug concentration.

[0034] (7) During the entire experiment, pay attention to the changes in cell morphology.

[0035] According to the results of cytopathology (CPE) observation and MTT cytotoxicity assay, the toxicity of Saxifraga water extract to cells is significantly correlated with drug concentration, and increasing drug concentration will reduce cell activity. The lowest drug dilution concentration with a cell activity rate of more than 90% is taken as the highest safe drug concentration, and the results show that the safe concentration of Saxifraga water extract is 4.88 mg / mL.

[0036] 2. Drug anti-PRRSV activity test

[0037] About 5×104 cells were added to each well of a 96-well plate and placed in a 37°C incubator for 2-3 hours. After removing the culture medium, PRRSV virus solution (infection dose was 0.5 MOI) was added. After 2 hours of virus infection, the virus solution was removed and replaced with 200 μL / well of fresh culture medium with the highest safe concentration of the drug. Cell fluids at different time points (12, 24, 36 and 48 hours) after virus infection in each experimental group were collected for RNA extraction, and the absolute quantitative fluorescence PCR (RT-qPCR) method was used to determine the PRRSV N gene copy number.

[0038] Determination of the anti-PRRSV activity of various Chinese herbal medicine extracts Figure 1 As shown. Among them, Figure 1 The control group and groups 1-10 were the PRRSV-infected group without drug addition, the alcohol-extracted crab group, the water-extracted Achyranthes bidentata group, the water-extracted Codonopsis pilosula group, the water-extracted Saxifraga heracleifolia group, the water-extracted Senecio chinensis group, the water-extracted Pinus armeniaca group, the water-extracted Atractylodes macrocephala leaf group, the water-extracted Rubia cordifolia group, the alcohol-extracted Phyllanthus emblica group and the water-extracted Meconopsis pilosula group.

[0039] Depend on Figure 1 It can be seen that compared with the PRRSV N gene copy number of the control group, the water extract of Saxifraga herba (4.88mg / mL) has a very significant difference on PRRSV (p<0.001); while the other Chinese herbal medicine extracts have no significant difference on the PRRSV N gene copy number compared with the control group (p>0.05). The results show that the water extract of Saxifraga herba has a significant inhibitory effect on the in vitro replication of PRRSV.

[0040] 3. Indirect Immunofluorescence (IFA) Assay

[0041] Indirect immunofluorescence assay (IFA) was used to detect the virus infection of PAM cells at different drug concentrations. The PRRSV N protein showed green fluorescence under fluorescence, and the cell nucleus showed blue fluorescence after DAPI staining. The superposition of the two can show the location of cell infection. Cell infection rate = number of infected cells / total number of cells 100%.

[0042] The results of the IFA test of HEC against PRRSV are as follows Figure 2As shown in the figure, N protein: PRRSV N protein monoclonal antibody IFA identification, the secondary antibody is goat anti-rabbit FITC (green fluorescence); DAPI: cell nucleus DAPI staining (blue); Merge: N protein and DAPI fusion map; "+" indicates PRRSV infection (0.5MOI); "-" indicates no PRRSV infection.

[0043] Depend on Figure 2 It can be seen that the cells in the double negative group (no drug and PRRSV added) had no obvious fluorescence except for a small amount of dye residue. The cells in the PRRSV infection group (0μg / mL drug) showed a large amount of green fluorescence signal of N protein. When the drug concentration was 1.22mg / mL, a small amount of green fluorescence signal was presented. As the drug concentration increased, the green fluorescence signal gradually decreased, indicating that the number of infected cells decreased with the increase of drug concentration. The cell nucleus showed a uniform blue fluorescence signal after DAPI staining, and the cells in the 0μg / mL drug group in the PRRSV infection group showed obvious cell aggregation. When the drug concentration was 1.22, 2.44 and 4.88mg / mL, the cell infection rate was significantly lower than that of the positive control group (p<0.001). The results showed that the drug concentration was 1.22, 2.44 and 4.88mg / mL, which had a significant inhibitory effect on PRRSV.

[0044] 4. Evaluation of the antiviral activity of drugs against new PRRSV strains

[0045] About 5×104 cells were added to each well of a 96-well plate, and the plate was placed in a 37°C incubator for 2-3 hours. After the culture medium was removed, 100 μL / well of the latest popular PRRSV-2 strain virus solution (infection dose was 0.5 MOI) was added. After 2 hours of virus infection, the virus solution was removed and replaced with 200 μL / well of fresh culture medium with different drug concentrations. IFA was used to detect the infection of PAM with different drug concentrations, and the cell infection rate was calculated.

[0046] The drug concentration of Saxifraga herba water extract (HEC) was selected as 4.88 mg / mL, and the antiviral activity of the drug against three different lineages of virus strains (SCwhn09CD, SCN17 and GZgy17) was evaluated by RT-qPCR and IFA methods. Figure 3 As shown. Among them, Figure 3 In the figure, (a) PRRSV N gene copy number determination; (b) PAM cell infection rate determination; *** indicates extremely significant difference (p<0.001); ns indicates no significant difference (p>0.05).

[0047] Depend on Figure 3It can be seen that compared with the positive control group (PRRSV infection group without drug addition), the N gene copy number of the saxifrage water extract test group was significantly lower than that of the positive control group (p<0.001), while there was no significant difference in the N gene copy number among the three strains (p>0.05). In addition, the cell infection rate of the saxifrage water extract test group was significantly lower than that of the positive control group (p<0.001), while there was no significant difference in the cell infection rate of the three different PRRSV strains (p>0.05), indicating that the saxifrage water extract has obvious anti-PRRSV activity against different lineages of PRRSV strains.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of a water extract of Saxifraga as the sole active ingredient in the preparation of an anti-porcine reproductive and respiratory syndrome virus drug, characterized in that: The saxifrage water extract is prepared by the following method: 100 g of saxifrage powder is wrapped with filter paper and then placed in a Soxhlet extractor, and neutral deionized water with a mass of 10 times that of the saxifrage powder is added to extract for 5 hours, and after the water extract is recovered, it is concentrated and fixed to volume in a water bath at 37° C. After the fixed volume, the concentration of saxifrage in the concentrated solution is 5 g / mL, and the saxifrage water extract is obtained.

2. An anti-porcine reproductive and respiratory syndrome virus drug, characterized in that: The invention comprises the water extract of Saxifraga saxifrage as claimed in claim 1 as the only active ingredient.

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