Use of a salt bush extract in the preparation of a medicine against porcine epidemic diarrhea virus

Abstract

The application discloses application of a Halodendronn barosum extract in preparation of a medicine for resisting porcine epidemic diarrhea virus, and belongs to the technical field of plant extract antiviral technology. The Halodendronn barosum extract comprises a Halodendronn barosum water extract or a Halodendronn barosum alcohol extract. It is verified through experiments that the 10-80 mg / mL Halodendronn barosum water extract has no obvious toxicity to Vero cells, can inhibit proliferation of PEDV in a dose-dependent manner, and has a better antiviral effect in vitro than the alcohol extract; and the medicine is safe in vivo. The Halodendronn barosum extract can effectively improve clinical symptoms such as temperature disorder, growth obstruction and diarrhea of infected piglets, significantly reduce virus load in the intestinal tract, and reduce pathological damage to intestinal tissue. In summary, the Halodendronn barosum extract has good biological safety and in-vivo and in-vitro anti-PEDV activity, meets the requirements of green veterinary medicine research and development, and has high industrialization development and clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of antiviral technology of plant extracts, and in particular to the application of *Salvia splendens* extract in the preparation of drugs against porcine epidemic diarrhea virus. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae and the genus Alphacoronavirus. PEDV is an enteropathogenic coronavirus that causes symptoms such as diarrhea and vomiting in pigs, with particularly high mortality rates in newborn piglets, resulting in significant economic losses to the pig industry. Currently, there is a lack of effective vaccines and specific drugs for PEDV, and the emergence of mutant strains with stronger virulence, infectivity, and pathogenicity poses a significant challenge to global PEDV control, necessitating the development of safe and effective vaccines and antiviral drugs. In recent years, PEDV has exhibited a pattern of sporadic outbreaks, localized outbreaks, recurrent infections, and difficulty in eradication, causing severe losses to large-scale pig farms, highlighting the urgent need for efficient and safe control measures.

[0003] Current PEDV control primarily relies on vaccination, chemical drugs, and biosafety. Regarding vaccines, due to the high frequency of PEDV mutations, the matching degree between existing strains and prevalent field strains has decreased, leading to significant problems such as immunization failure and insufficient protection rates. These issues, coupled with maternal antibody interference and non-standard immunization procedures, make effective blocking difficult. Chemical antiviral drugs, on the other hand, suffer from drawbacks such as frequent drug resistance, drug residues, toxic side effects, and disruption of the gut microbiota, limiting their application under national policies promoting antibiotic reduction and green farming. Therefore, developing low-toxicity, residue-free, and non-resistant natural antiviral drugs has become a core direction of veterinary pharmacology research and a necessity for the industry.

[0004] Desert halophytes are a unique medicinal resource in my country, characterized by strong resilience and abundant secondary metabolites. They are rich in active ingredients such as flavonoids, polyphenols, terpenes, alkaloids, and polysaccharides, possessing multiple activities including anti-inflammatory, antioxidant, antiviral, and immunomodulatory effects, making them a high-quality raw material source for developing green veterinary drugs. *Salmonella halophyte*, a dominant halophyte shrub in Xinjiang, is widely distributed, abundant, and has strong regeneration capabilities. It does not occupy arable land and possesses both ecological and economic value. Currently, there are no reports of its extracts exhibiting anti-PEDV activity. Summary of the Invention

[0005] The purpose of this invention is to provide the application of *Salvia splendens* extract in the preparation of drugs against porcine epidemic diarrhea virus (PEDV), thereby solving the problems existing in the prior art. *Salvia splendens* extract has both good biocompatibility and in vitro and in vivo anti-PEDV activity, meets the requirements of green veterinary drug development, and has high potential for industrialization and clinical application.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of *Salvia splendens* extract in the preparation of drugs against porcine epidemic diarrhea virus, wherein the *Salvia splendens* extract includes *Salvia splendens* aqueous extract or *Salvia splendens* alcoholic extract.

[0007] Optionally, the water extract of *Salvia splendens* is obtained by soaking pulverized *Salvia splendens* powder in water, decocting it three times, combining the filtrates, concentrating, and sterilizing under high pressure.

[0008] Optionally, the aqueous extract of *Salvia splendens* is prepared by the following method: *Salvia splendens* is pulverized to obtain *Salvia splendens* powder; the *Salvia splendens* powder is soaked in 10 times its volume of pure water for 6 hours, decocted for 3 hours, filtered, and the first filtrate and the first residue are collected; the first residue is decocted in 10 times its volume of pure water for 3 hours, filtered, and the second filtrate and the second residue are collected; the second residue is decocted in 10 times its volume of pure water for 3 hours, filtered, and the third filtrate is collected; the first filtrate, the second filtrate, and the third filtrate are combined, concentrated, and autoclaved.

[0009] Optionally, the ethanol extract of *Salvia splendens* is obtained by soaking pulverized *Salvia splendens* powder in ethanol, ultrasonically extracting it three times, combining the filtrates, concentrating, and autoclaving.

[0010] Optionally, the *Salvia splendens* alcohol extract is prepared by the following method: *Salvia splendens* is pulverized to obtain *Salvia splendens* powder; the *Salvia splendens* powder is soaked in 10 times its volume of 70% ethanol for 48 h, sonicated at 30°C for 30 min, filtered, and the first filtrate and the first filter residue are collected; the first filter residue is soaked in 10 times its volume of 70% ethanol at 30°C for 30 min, filtered, and the second filtrate and the second filter residue are collected; the second filter residue is soaked in 10 times its volume of 70% ethanol at 30°C for 30 min, filtered, and the third filtrate is collected; the first filtrate, the second filtrate, and the third filtrate are combined, concentrated, and autoclaved.

[0011] Optionally, the *Salvia splendens* extract is a water extract of *Salvia splendens*.

[0012] Optionally, the *Salvia splendens* extract can reduce the viral load of porcine epidemic diarrhea virus in the intestine.

[0013] Optionally, the *Salvia splendens* extract can alleviate intestinal pathological damage caused by porcine epidemic diarrhea virus.

[0014] The present invention discloses the following technical effects: This invention uses *Salvia splendens*, a halophyte native to Xinjiang, as raw material. Extracts were prepared using both water decoction and ultrasonic extraction with 70% ethanol. In vitro cytotoxicity, in vitro anti-PEDV activity, in vivo safety in piglets, and in vivo anti-PEDV efficacy tests were systematically conducted to comprehensively evaluate the stability of the extraction process, drug safety, and antiviral activity. Results showed that the 10–80 mg / mL *Salvia splendens* water extract had no significant toxicity to Vero cells and could inhibit PEDV proliferation in a dose-dependent manner, exhibiting better in vitro antiviral effects than the ethanol extract. A 14-day in vivo safety test showed that 10 times the clinical therapeutic dose of *Salvia splendens* extract had no adverse effects on piglet body temperature, weight, or physiological status, indicating safe in vivo administration. Animal challenge experiments with the PEDV-G2c strain (CHjx20250) confirmed that *Salvia splendens* extract could effectively improve clinical symptoms such as fever disturbances, growth retardation, and diarrhea in infected piglets, significantly reduce viral load in the intestines, and alleviate pathological damage to intestinal tissues. In summary, the extract of *Salvia splendens* exhibits both good biocompatibility and in vitro and in vivo anti-PEDV activity, meeting the requirements for green veterinary drug development and possessing high potential for industrialization and clinical application. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The cytotoxicity of *Salvia spicatum* extract to Vero; Figure 2 Fluorescence spectrum (A) and antiviral effect (B) of different extracts of *Salix matsudana* against PEDV (DR13). Figure 3 In vitro resistance to PEDV in water-extracted samples of *Salix babylonica*; Figure 4 Changes in body temperature (A) and body weight (B) in piglets under 10 times the dose of *Salvia splendens* water extract; Figure 5 The results of the in vivo test of *Salix babylonica* against PEDV are as follows: A: Piglet survival curve; B: Piglet body temperature change; C: Piglet weight change; D: Piglet enteric virus load. Figure 6 The results of the necropsy of piglets; Figure 7 The results of immunohistochemistry of the piglet intestine. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Example 1 1. Materials and Instruments Experimental materials: dried *Salix babylonica* plants; purified water, 70% ethanol (v / v); DMEM high-glucose medium, fetal bovine serum, CCK-8 kit, PBS buffer; DR13 (PEDV-DR13-GFP), HB (GIIb), HW (GIIa), CV777 (GIa), and CHjx2025 (GIIc) strains (all strains were provided by Professor Li Yongtao of Henan Agricultural University and are publicly available in the literature https: / / doi.org / 10.1016 / j.virol.2023.109877 and DOI: 10.1186 / S12917-026-05714-2), PEDV nucleic acid detection kit; physiological saline, sterilization and disinfection reagents, etc.

[0023] Experimental animals and cells: Vero cells were used; healthy (without common diarrhea pathogens) 3-day-old piglets with similar weight and age were selected and kept in isolation in single pens during the experiment.

[0024] Main instruments: grinder, electronic balance, ultrasonic cleaner, vacuum filtration device, rotary evaporator, high-pressure steam sterilizer, CO2 incubator, inverted fluorescence microscope, enzyme-linked immunosorbent assay (ELISA) reader, real-time fluorescence quantitative PCR instrument, veterinary thermometer, electronic weighing scale, piglet feeding cages, etc.

[0025] 2. Preparation of Salt Spike Wood Extract After crushing the saltwort, the mixture was divided into two equal portions, and a water decoction and a 70% ethanol extract were prepared respectively. Both were concentrated to 1 g / mL, sterilized at 121℃ for 20 min, and then stored at 4℃ for later use.

[0026] Decoction: Soak the pulverized *Salvia splendens* powder in 10 times its volume of pure water for 6 hours. Bring to a boil over high heat, then simmer over low heat for 3 hours. Filter and collect the first filtrate and the first residue. Add 10 times its volume of pure water to the first residue, bring to a boil over high heat, then simmer over low heat for 3 hours. Filter and collect the second filtrate and the second residue. Add 10 times its volume of pure water to the second residue, bring to a boil over high heat, then simmer over low heat for 3 hours. Filter and collect the third filtrate (a total of 3 decoctions). Combine the three filtrates and concentrate to 1 g / mL. Alcoholic extract: The pulverized *Salvia splendens* powder was soaked in 10 times its volume of 70% ethanol for 48 h, sonicated at 30℃ for 30 min, filtered, and the first filtrate and the first residue were collected; the first residue was soaked in 10 times its volume of 70% ethanol, sonicated at 30℃ for 30 min, filtered, and the second filtrate and the second residue were collected; the second residue was soaked in 10 times its volume of 70% ethanol, sonicated at 30℃ for 30 min, filtered, and the third filtrate was collected (a total of 3 sonic extractions were performed). The three filtrates were combined and concentrated to 1 g / mL.

[0027] 3. Vero cell safety test of *Salix babylonica* extract Vero cells were cultured in DMEM medium containing 10% fetal bovine serum and penicillin-streptomycin at 37°C and 5% CO2. The experiment was conducted when the cells reached 80%–90% confluence. Extract concentration gradients were set up: 0, 10, 20, 40, 80, 160, and 320 mg / mL, with three replicates for each concentration and a blank control group. After 24 h of culture, the absorbance at 450 nm was measured using the CCK-8 assay to calculate cell viability and determine the safe effective concentration.

[0028] 4. In vitro anti-PEDV test of different *Salix salina* extracts A blank control group, a virus model group, and a drug intervention group were set up. In the drug intervention group, cells were pretreated with 20, 40, and 80 mg / mL water extract and ethanol extract, respectively, for 6 h. After washing with PBS, the cells were inoculated with DR13 virus strain at MOI=0.1. After virus adsorption for 1 h, the culture medium was replaced with drug-containing maintenance medium. After 12 h of culture, cytopathic effects and green fluorescent protein (GFP) expression were observed. The supernatant was collected after three freeze-thaw cycles to detect virus titers, and the antiviral effects of the two types of extracts were compared.

[0029] 5. In vitro anti-PEDV test of water extracts from *Salvia spicatus* against different PEDVs A blank control group, a virus model group, and a drug intervention group were set up. In the drug intervention group, cells were pretreated with 20, 40, and 80 mg / mL water extract for 6 h, respectively. After washing with PBS, cells were inoculated with DR13, HB (GIIb), HW (GIIa), CV777 (GIa), and CHjx2025 (GIIc) strains at MOI=0.1, respectively. The drug-containing maintenance medium was replaced 1 h after virus adsorption. After 12 h of culture, cytopathic effects, green fluorescent protein (GFP) expression (DR13), and immunofluorescence (HB, HW, CV777, and CHjx2025) were observed to compare the effects of the water extract against different viral genotypes.

[0030] 6. In vivo safety test in piglets Eight piglets free of specific pathogens and antibodies were randomly divided into a PBS control group and an extract treatment group, with four piglets in each group. The treatment group was administered 10 times the clinical therapeutic dose of *Salvia splendens* extract by gavage daily, while the control group was given an equal volume of physiological saline. The treatment was continued for 14 days. The piglets' body temperature and weight were monitored daily, and their feed intake, mental state, and fecal condition were observed and recorded.

[0031] 7. In vivo efficacy test of anti-PEDV drugs in piglets Twelve piglets without specific pathogens or antibodies were selected and divided into a blank control group, a PEDV infection model group (challenge control), and a *Salix matsudana* treatment group, with four piglets in each group. All piglets were kept in isolation throughout the experiment. On day 3 of the experiment, the model group and the treatment group were orally inoculated with the CHjx2025 (GIIc) strain (10... 6 TCID 50 / head); the treatment group received water extract by gavage (2 mL / time, twice a day) for 6 consecutive days starting 3 days before viral challenge. Body temperature, weight, morbidity, and mortality were recorded daily. On day 5 of the experiment, piglets were euthanized, and duodenal, jejunal, and ileal tissues were collected. Viral load was detected by RT-qPCR. Histopathological damage was observed through gross necropsy and immunohistochemistry of intestinal tissues (jejunum and ileum).

[0032] 8. TCID 50 Measurement Virus solution was serially diluted 10-fold and then used to infect monolayer Vero cells. Each dilution was performed in 6 replicates. Cytopathic effects were recorded after 96 hours of continuous observation. TCID was calculated using the Reed-Muench method. 50 .

[0033] 9. Total RNA extraction and RT-qPCR Total RNA was extracted from the samples using TRNzol reagent, and the RNA concentration was determined using an ultra-micro spectrophotometer. cDNA was synthesized using the accompanying kit, and quantitative real-time PCR was performed to detect the PEDV gene copy number. PEDV-RdRp detection primers: upstream primer: 5′-CCGAACACGATTTCTTCACT-3′ (SEQ ID NO.1), downstream primer: 5′-ACTCCTCACAAGCACCTACCT-3′ (SEQ ID NO.2).

[0034] 10. Immunofluorescence (IFA) Detection Method Immunofluorescence was used to observe PEDV replication in cells. First, the cell culture medium was discarded, and the cells were gently washed three times with PBS. Then, 4% paraformaldehyde (PFA, Solarbio, Beijing, China) was added for fixation at room temperature for 15 min. After fixation, the cells were washed three more times with PBS to remove residual fixative. Mouse anti-PEDV S protein monoclonal antibody (1:1000 dilution) prepared in our laboratory was added to the culture wells, and the cells were incubated at 37°C for 1 h. After incubation, the cells were washed three times with PBS, and then FITC-labeled goat anti-mouse IgG (H+L) secondary antibody (1:500 dilution, Wuhan Proteintech) was added, and the cells were incubated at 37°C in the dark for 1 h. After discarding the secondary antibody and washing with PBS, the cell nuclei were stained with DAPI staining reagent (Solarbio, Beijing, China). Finally, the cell fluorescence images were observed and acquired using a laser confocal microscope (Zeiss LSM 800, Oberkochen, Germany).

[0035] 11. Statistical Analysis Data were analyzed using GraphPad Prism 8.0.2 software. One-way ANOVA or independent samples t-tests were used for comparisons between groups. Results are expressed as mean ± standard deviation, and each trial was conducted in triplicate. A p-value < 0.05 was considered statistically significant.

[0036] 12. Experimental Results 12.1 Evaluation of the anti-PEDV effect of *Salvia splendens* extract First, the cytotoxicity of different concentrations of *Salvia splendens* extract on Vero cells was detected using a CCK-8 assay. The results showed that the aqueous and alcoholic extracts at concentrations of 10–80 mg / mL had no significant cytotoxicity to the cells, with cell viability approaching 100%; however, significant cytotoxicity was observed at concentrations ≥160 mg / mL. Based on these results, the safe in vitro concentration range was determined. Figure 1 ).

[0037] In vitro antiviral assays were conducted using aqueous and ethanolic extracts at concentrations of 20, 40, and 80 mg / mL. Cells were pretreated with PEDV-DR13-GFP before inoculation with the virus strain. Results showed that the extracts alleviated cytopathic effects and reduced viral fluorescence expression in a dose-dependent manner, with the aqueous extract exhibiting superior antiviral activity compared to the ethanolic extract. Figure 2 ).

[0038] Cells were pretreated with 20, 40, and 80 mg / mL of *Salvia splendens* aqueous extract, and then inoculated with DR13, HB, HW, CV777, and CHjx2025 strains, respectively. Results showed that the aqueous extract dose-dependently reduced cytopathic effects and viral fluorescence signal, and exhibited significant inhibitory effects against multiple strains. Figure 3 ).

[0039] 12.2 Safety test results of *Salvia spicatus* extract in piglets Continuous monitoring over 14 days revealed that piglets in both the treatment group and the control group had normal body temperature and weight, and no significant difference in growth status. This indicates that 10 times the clinical dose of *Salvia splendens* extract has no acute toxicity to piglets and is safe for in vivo administration. Figure 4 ).

[0040] 12.3 Results of in vivo anti-PEDV efficacy experiment in piglets During the experiment, piglets in the control group showed no abnormalities; all piglets in the PEDV model group exhibited typical symptoms such as diarrhea, lethargy, fever, and significant weight loss. The number of affected piglets in the *Salvia splendens* treatment group decreased, and the abnormalities in body temperature and weight were significantly improved; no piglets died throughout the entire trial. Figure 5 AC (Chinese)

[0041] On day 5 of the experiment, piglets were necropsed and their enteric viral load was measured: PEDV was not detected in the intestines of the control group; the viral load in the jejunum and ileum of the model group was 10. 7.9 and 10 6.3 The viral load in the corresponding intestinal segment of the group treated with *Salix matsudana* extract decreased to 10. 5.9 and 10 5.0 ( Figure 5 (D).

[0042] Gross necropsy revealed that the intestinal walls of piglets in the PEDV-infected group were significantly thinner, and they experienced severe diarrhea throughout the course of the disease; although piglets in the Salt-Spike Wood-treated group initially experienced diarrhea, the diarrhea symptoms completely subsided after 3 days of administration, and the intestinal tissue morphology was more similar to that of the blank control group. Figure 6 Immunohistochemical observation further confirmed that the PEDV-challenged group showed significant villus loss in the intestinal tissue, while the intestinal tissue structure of the *Salvia splendens* treatment group remained intact, showing no significant abnormalities compared to the blank control group. Figure 7 ).

[0043] In summary, in vitro experiments showed that the *Salvia splendens* extract at a concentration of 10-80 mg / mL is a safe concentration and can dose-dependently inhibit PEDV proliferation in Vero cells, demonstrating significant in vitro antiviral effects. In vivo experiments showed that the *Salvia splendens* extract has no acute toxicity and does not affect the body temperature, growth, or normal physiological state of piglets, indicating its safety for in vivo administration. PEDV challenge animal experiments confirmed that the *Salvia splendens* extract can effectively alleviate the symptoms of PEDV infection in piglets, correct abnormal body temperature, and improve growth inhibition, demonstrating good in vivo prevention and treatment effects against PEDV infection. In conclusion, the *Salvia splendens* extract is safe and effective, and is a promising natural anti-PEDV veterinary raw material that can provide new ideas and experimental support for the high-value utilization of halophytes in Xinjiang and the green prevention and control of PEDV in pigs.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of *Salvia spicatum* extract in the preparation of drugs against porcine epidemic diarrhea virus, characterized in that, The *Salvia splendens* extract includes either a water extract or an alcohol extract of *Salvia splendens*.

2. The application as described in claim 1, characterized in that, The aqueous extract of *Salvia splendens* is obtained by soaking pulverized *Salvia splendens* powder in water, decocting it three times, combining the filtrates, concentrating, and sterilizing under high pressure.

3. The application as described in claim 2, characterized in that, The aqueous extract of *Salvia splendens* is prepared by the following method: *Salvia splendens* is pulverized to obtain *Salvia splendens* powder. The *Salvia splendens* powder is soaked in 10 times its volume of pure water for 6 hours, decocted for 3 hours, filtered, and the first filtrate and the first residue are collected. The first residue is decocted in 10 times its volume of pure water for 3 hours, filtered, and the second filtrate and the second residue are collected. The second residue is decocted in 10 times its volume of pure water for 3 hours, filtered, and the third filtrate is collected. The first filtrate, the second filtrate, and the third filtrate are combined, concentrated, and autoclaved.

4. The application as described in claim 1, characterized in that, The ethanol extract of *Salix babylonica* was obtained by soaking pulverized *Salix babylonica* powder in ethanol, ultrasonically extracting it three times, combining the filtrates, concentrating, and autoclaving.

5. The application as described in claim 4, characterized in that, The *Salvia splendens* alcohol extract was prepared by the following method: *Salvia splendens* was pulverized to obtain *Salvia splendens* powder; the *Salvia splendens* powder was soaked in 10 times its volume of 70% ethanol for 48 h, sonicated at 30°C for 30 min, filtered, and the first filtrate and the first filter residue were collected; the first filter residue was soaked in 10 times its volume of 70% ethanol at 30°C for 30 min, filtered, and the second filtrate and the second filter residue were collected; the second filter residue was soaked in 10 times its volume of 70% ethanol at 30°C for 30 min, filtered, and the third filtrate was collected; the first filtrate, the second filtrate, and the third filtrate were combined, concentrated, and autoclaved.

6. The application as described in claim 1, characterized in that, The extract of *Salvia splendens* is a water extract of *Salvia splendens*.

7. The application as described in claim 1, characterized in that, The extract of *Salvia spicatum* can reduce the viral load of porcine epidemic diarrhea virus in the intestine.

8. The application as described in claim 1, characterized in that, The extract of *Salvia spicatus* can alleviate intestinal pathological damage caused by porcine epidemic diarrhea virus.

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