Application of Baicalein Combined with Gemcitabine in the Preparation of Drugs against Porcine Epidemic Diarrhea Virus

Abstract

The present invention discloses the application of baicalein in combination with gemcitabine in the preparation of drugs against porcine epidemic diarrhea virus. For the first time, it is verified that both baicalein and gemcitabine have certain preventive and therapeutic effects on porcine epidemic diarrhea. By comprehensively using cell biology and virology techniques, it is first discovered that the combined use of baicalein and gemcitabine can inhibit the in vitro infection of PEDV, providing another combined drug use scheme for antiviral drug combinations against PEDV. The present invention proves through in vitro cell experiments that the combination of baicalein and gemcitabine inhibits the proliferation of PEDV in cells, and the effect is significantly higher than that of single drug use. Moreover, good therapeutic effects can be achieved at low doses, with significant synergistic effects, and advantages such as relatively low toxicity and high safety concentration. It has very good application prospects in the preparation of drugs for preventing and / or treating porcine epidemic diarrhea virus.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for synergistic antiviral effects. Specifically, the pharmaceutical composition contains the natural product baicalein and the nucleoside analog gemcitabine. Background Art

[0002] Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an economically important intestinal disease that seriously affects the pig farming industry. Clinically, once neonatal piglets are infected with PEDV, they often show severe diarrhea, vomiting, dehydration, weight loss and other symptoms, and the mortality rate is as high as over 80%; although adult pigs do not show a high mortality rate after being infected with PEDV, they will also have watery diarrhea and loss of appetite after infection, which affects the breeding efficiency; in addition, after lactating sows are infected with PEDV, they will have no milk, which affects the rearing of piglets, and PEDV will also damage the reproductive function of adult pigs, affecting the breeding of seedlings and the development of the pig farming industry.

[0003] Studies have found that PED may occur throughout the year, and PEDV has a strong infectivity and a short latency period. Clinical symptoms will appear about 2 days after the pig herd is infected; the virus will also spread to the entire pig herd in a short time, resulting in a long course of disease and great difficulty in prevention and control. In addition, the occurrence of this disease is also cyclical. Pig farms that have had the disease before will experience repeated outbreaks of the epidemic in the pig farm due to the PEDV surviving in the environment. These characteristics make the epidemic in pig farms last for a long time, resulting in a state where some pig farms have no piglets to raise, bringing great troubles and serious economic losses to the pig farming industry.

[0004] Currently, the prevention of porcine epidemic diarrhea mainly relies on PED vaccines. There are many existing PED-related vaccines, including inactivated vaccines, attenuated live vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle (VLP) vaccines, live vector vaccines, etc. However, PEDV is an RNA virus belonging to the genus Alphacoronavirus of the family Coronaviridae, with a high variability. It is necessary to continuously update the vaccine strains to match the prevalent strains, and the immune effect and safety of the vaccines also need to be carefully verified. The safety and potency of the vaccines often fail to achieve the prevention and control effect. In the treatment of PED, generally, symptomatic treatment of diseased pigs at different stages is carried out by means such as fluid replacement, anti-inflammatory, prevention of dehydration, and avoidance of secondary infection. A large amount of antibiotics are often used, and it is difficult to effectively inhibit the proliferation of PEDV in cells and control the outbreak of PED; it is necessary to control the spread of PEDV by means such as isolation, cleaning the pen, and purification. The treatment effect is lagging, the prevention and control measures are complex, and the effect is not good. Therefore, exploring potential small molecule inhibitors against PEDV may be a breakthrough in the prevention and control of PED.

[0005] Baicalein is a flavonoid compound extracted from the traditional Chinese medicine Scutellaria baicalensis Georgi. In Compendium of Materia Medica, Scutellaria baicalensis Georgi is used to treat wind-heat and damp-heat, and lung atrophy with fishy throat odor. It is generally considered to have pharmacological effects such as anti-inflammatory, antioxidant, anti-tumor, and anti-microbial activities. In the prior art, Chinese patent document CN116019805B discloses the application of wogonin in anti-porcine epidemic diarrhea virus, revealing that wogonin can significantly inhibit the internalization, intracellular replication, and release of new mature virus particles of porcine epidemic diarrhea virus by targeting porcine epidemic diarrhea virus-3C-like protease (PEDV-3CLpro), and can also directly inactivate porcine epidemic diarrhea virus in vitro. However, wogonin and baicalein have different chemical structures (as Figure 1 shown) and pharmacological effects. Baicalein is a 5,6,7-trihydroxyflavone, while wogonin is 5,7-dihydroxy-8-methoxyflavone. Baicalein has the effects of reducing cerebrovascular resistance, improving cerebral blood circulation, increasing cerebral blood flow, and anti-platelet aggregation, and is clinically used for the treatment of paralysis after cerebrovascular diseases. Wogonin, on the other hand, has spasmolytic, anti-cancer, and diuretic effects. Therefore, although wogonin has the functions of inhibiting and inactivating porcine epidemic diarrhea virus, whether baicalein has the same efficacy and anti-virus mechanism is unknown.

[0006] Gemcitabine is an analogue of cytosine nucleotide, and its structure is as Figure 2 shown. It is an effective anti-tumor drug and plays an important role in the treatment of various cancers. Clinically, it can be applied to the treatment of advanced pancreatic cancer, locally advanced and metastatic stage IV non-small cell cancer. In the prior art, the use of nucleoside analogues for the prevention and treatment of porcine epidemic diarrhea is rarely reported. Chinese patent document CN115448924B discloses the application of nucleoside analogues or combined preparations containing nucleoside analogues in anti-virus, involving that nucleoside analogues can be used as inhibitors of porcine epidemic diarrhea virus (PEDV) replication. However, the nucleoside analogues disclosed therein do not include gemcitabine. Whether it also has anti-porcine epidemic diarrhea virus as an antimetabolic chemotherapy drug has not been reported.

[0007] In addition, Chinese patent document CN113662953B discloses the application of vidarabine in the preparation of a drug resistance sensitizer for anti-tumor chemotherapy drugs. The antiviral drug vidarabine is combined with the anti-tumor drug gemcitabine. Vidarabine is a sensitizer or drug resistance reverser for anti-tumor drugs to effectively kill tumor cells, providing a new way and means for the effective treatment of tumors. However, the combined use of baicalein and gemcitabine to prevent and / or treat porcine epidemic diarrhea virus infection has not been reported, and their combined effect also urgently needs to be studied and verified. Summary of the Invention

[0008] Regarding the issues of whether baicalein and gemcitabine mentioned above have the efficacy of preventing and treating porcine epidemic diarrhea, and how the combined use of baicalein and gemcitabine affects the prevention and treatment of porcine epidemic diarrhea, the present invention provides the application of the combination of baicalein and gemcitabine in the preparation of drugs against porcine epidemic diarrhea virus. It is verified for the first time that the combination of baicalein and gemcitabine has good preventive and therapeutic efficacy against porcine epidemic diarrhea. The combined use of the two can achieve the effect of inhibiting the in vitro infection of PEDV, providing another combination drug use plan for the antiviral drug combination of PEDV.

[0009] One of the purposes of the present invention is to provide the application of the combination of baicalein and gemcitabine in the preparation of drugs against porcine epidemic diarrhea virus.

[0010] Preferably, in the combined drug, the molar ratio of the dosage of baicalein to gemcitabine is 0.75 - 3:0.5 - 2. Experiments show that when the dosages of baicalein and gemcitabine are 0.75 μmol / L and 2 μmol / L respectively, the inhibition rate of the combination against PEDV can reach 54%, and the inhibition effect is better than that of the two compounds used alone, and the use concentration is lower than each EC50 value (the EC50 of gemcitabine is 3.14 μmol / L, and the EC50 of baicalein is 5.01 μmol / L). And when the concentration of gemcitabine is 0.5 μM and the concentration of baicalein is 0.75 μM, the synergistic effect of the two is optimal.

[0011] In addition, another purpose of the present invention is to provide a pharmaceutical composition for preventing and / or treating porcine epidemic diarrhea virus infection, which uses the combined drug of baicalein and gemcitabine or its pharmaceutically acceptable salt as the sole active ingredient or one of the active ingredients.

[0012] Advantages of the present invention:

[0013] 1) The present invention verifies for the first time that the combination of baicalein and gemcitabine has good preventive and therapeutic efficacy against porcine epidemic diarrhea; comprehensively utilizes cell biology and virology techniques, and discovers for the first time that the combined use of baicalein and gemcitabine can achieve the effect of inhibiting the in vitro infection of PEDV, providing another combination drug use plan for the antiviral drug combination of PEDV.

[0014] 2) The present invention reveals for the first time through cell proliferation cycle experiments that baicalein can inhibit the entry stage of PEDV-infected cells, while gemcitabine acts on inhibiting the replication stage of PEDV; the combined use of the two compounds acts on different stages of PEDV proliferation to exert a synergistic antiviral effect.

[0015] 3) The present invention proves through in vitro cell experiments that Baicalein combined with Gemcitabine inhibits the proliferation of PEDV in cells, and the effect is significantly higher than that of using the drugs alone. The experiments show that when the dosages of Baicalein and Gemcitabine are 0.75 μmol / L and 2 μmol / L respectively, the combined use can achieve an inhibition rate of 54% against PEDV. The inhibitory effects are better than those of the two compounds used alone, and the used concentrations are lower than the respective EC50 values (the EC50 of Gemcitabine is 3.14 μmol / L, and the EC50 of Baicalein is 5.01 μmol / L). Moreover, when the concentration of Gemcitabine is 0.5 μM and the concentration of Baicalein is 0.75 μM, the synergistic effect between the two is optimal. It shows that the combined use of the two drugs can achieve good therapeutic effects at low doses and has a significant synergistic effect.

[0016] 4) The present invention also finds that the combination of Baicalein and Gemcitabine not only has good curative effects, but also has the advantages of relatively low toxicity and high safety concentration, and has very good application prospects in the preparation of drugs for preventing and / or treating porcine epidemic diarrhea virus. Description of the Drawings

[0017] Figure 1 Chemical structural formulas of Baicalein and Wogonin;

[0018] Figure 2 Chemical structural formula of Gemcitabine;

[0019] Figure 3 Schematic diagram of the toxicity of Baicalein to Vero cells;

[0020] Figure 4 Schematic diagram of the toxicity of Gemcitabine to Vero cells;

[0021] Figure 5 Schematic diagram of the inhibitory effect of treating PEDV-infected Vero cells with different concentrations of Baicalein;

[0022] Figure 6 Schematic diagram of the inhibitory effect of treating PEDV-infected Vero cells with different concentrations of Gemcitabine;

[0023] Figure 7 Schematic diagram of the relative RNA content of treating PEDV-infected Vero cells with Baicalein;

[0024] Figure 8 Schematic diagram of the relative RNA content of treating PEDV-infected Vero cells with Gemcitabine;

[0025] Figure 9Schematic diagram of virus titers of Vero cells infected with PEDV treated with different concentrations of baicalein;

[0026] Figure 10 Schematic diagram of virus titers of Vero cells infected with PEDV treated with different concentrations of gemcitabine;

[0027] Figure 11 Schematic diagram of virus titers at different stages of Vero cells infected with PEDV treated with baicalein;

[0028] Figure 12 Schematic diagram of virus titers at different stages of Vero cells infected with PEDV treated with gemcitabine;

[0029] Figure 13 Schematic diagram of virus titers of Vero cells infected with PEDV treated with a combination of baicalein and gemcitabine at different concentrations;

[0030] Figure 14 Inhibitory rate of the combination of baicalein and gemcitabine against PEDV;

[0031] Figure 15 Analysis of the ZIP synergy index of the combination of baicalein and gemcitabine (in the figure, -10 < synergy score < 10 indicates an additive effect; synergy score < -10 indicates an antagonistic effect; synergy score > 10 indicates a synergistic effect);

[0032] Figure 16 Schematic diagram of the inhibitory effect of the combination of baicalein and gemcitabine at different concentrations on Vero cells infected with PEDV;

[0033] Figure 17 Schematic diagram of the cytotoxicity of the combination of baicalein and gemcitabine at different concentrations on Vero cells. Detailed implementation mode

[0034] The following further details the specific implementation mode of the present invention in conjunction with embodiments. Reagents without a source provided in the following experiments are all commercially available reagents, and methods not described in detail are all conventional well-known experimental methods. Baicalein and gemcitabine involved in each embodiment have the following structural formulas:

[0035] (Baicalein);

[0036] (Gemcitabine).

[0037] The inhibitory effects of baicalein and gemcitabine on porcine deltacoronavirus (PDCoV), as well as the combined drug effects of the two, were verified in detail, demonstrating that the combination of baicalein and gemcitabine can be used as an active ingredient of a pharmaceutical composition for prevention and / or treatment purposes. The specific embodiments are as follows:

[0038] Example 1: Cytotoxicity identification of baicalein / gemcitabine on Vero cells

[0039] Vero cells were grown in an incubator at 37 °C with 5% CO2. After the cells in the culture flask were confluent, Vero cells were seeded in a 96-well cell culture plate at a density of 2.5×10 5 cells / mL, and 100 μL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well. The cells were cultured in an incubator at 37 °C with 5% CO2 for 24 h; the original cell culture medium was removed and replaced with DMEM medium containing different concentrations of baicalein / gemcitabine (baicalein: 0, 0.2, 0.4, 0.75, 1.5, 3.75, 6.25, 25, 50, 100, 200 μmol / L; gemcitabine: 0, 6.25, 12, 25, 50, 100, 200 μmol / L), which were serially diluted, and the control group was added with an equal volume of DMSO. After incubation at 37 °C for 24 h, 10 μL of CCK-8 reagent was added and the cells were cultured in an incubator at 37 °C with 5% CO2 for 2 h; cytotoxicity identification was performed according to the operation instructions.

[0040] The results are shown in Figure 3 and Figure 4 As shown, compared with the cell viability of the treatment group without baicalein / gemcitabine (DMSO group), the cell viability of the baicalein treatment group with a concentration between 0.2 μmol / L and 50 μmol / L was higher than 80%; when the concentration was between 100 μmol / L and 200 μmol / L, the cell viability was higher than 60%, indicating that the safe concentration of baicalein was less than 50 μmol / L. Therefore, a concentration of 10 μmol / L was selected for subsequent verification of the virus effectiveness; the cell viability of the gemcitabine treatment group with a concentration between 6.25 μmol / L and 200 μmol / L was higher than 80%, indicating that gemcitabine did not have strong cytotoxicity to Vero cells. A concentration of 10 μmol / L was selected as the highest effective concentration for gemcitabine antiviral verification.

[0041] Example 2: Verification of the inhibitory effect of baicalein / gemcitabine on Vero cell infection with PEDV

[0042] Vero cells were cultured according to the method of Example 1. After the cells grew to confluence, the cells were seeded at 2.5×10 5 cells / mL in a 24-well plate. 1 mL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well and cultured in an incubator at 37 °C and 5% CO2 for 24 h; the medium was replaced with fresh DMEM medium, and the Vero cells were infected with the PEDV AJ1102 strain at a multiplicity of infection (MOI) of 0.1 at 37 °C for 2 h; the cell supernatant was discarded, and the cells were washed three times with phosphate buffer (PBS) to wash away the virus particles that did not invade the cells. In the experimental group, media containing different concentrations of baicalein or gemcitabine (concentrations were 1 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, 10 μmol / L) were added, and in the control group, an equal volume of DMSO was added. After incubation for 16 h, the cells were fixed with 4% paraformaldehyde, incubated with PEDV N antibody at room temperature for 2 h, incubated with fluorescent secondary antibody in the dark for 1 h, incubated with DAPI for 15 min, and the results were observed under an inverted microscope.

[0043] The results are shown in Figure 5 and Figure 6 As shown. Compared with the control group (DMSO group), the green fluorescence expression levels in the 1 μmol / L baicalein treatment group were similar. Only a small amount of green fluorescence was observed in the 2 μmol / L baicalein treatment group. The green fluorescence expression disappeared in the 4 μmol / L - 20 μmol / L baicalein treatment groups, indicating that baicalein can significantly reduce the number of PEDV-infected Vero cells when the concentration is higher than 4 μmol / L, and the anti-PEDV proliferation is concentration-dependent at 4 μmol / L; the green fluorescence expression disappeared in the 10 μmol / L - 20 μmol / L gemcitabine treatment groups; green fluorescence expression was observed in the treatment groups with concentrations lower than 8 μmol / L, and the green fluorescence expression increased with the decrease in concentration, indicating that gemcitabine can significantly inhibit PEDV proliferation when the concentration is higher than 10 μmol / L, and the anti-PEDV proliferation is concentration-dependent at 10 μmol / L.

[0044] Example 3: Effect of Baicalein / Gemcitabine on the RNA Content of Vero Cells Infected with PEDV

[0045] Vero cells were seeded at 2.5×10 5The cells were plated with 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 24-well plate, and 1 mL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well, and cultured in a 37° C., 5% CO2 incubator for 24 h; a new DMEM medium was replaced, and the Vero cells were infected with the PEDV AJ1102 strain at a virus infection multiplicity (MOI) of 0.1 at 37° C. for 2 h; the cell supernatant was discarded, and the cells were washed three times with phosphate buffered saline (PBS) to wash away the virus particles that did not invade the cells, and a culture medium containing 1 μmol / L to 10 μmol / L baicalein / gemcitabine was added and incubated for 16 h (the concentration and control group settings were the same as in Example 2). The lysate was collected, and total RNA was extracted using the RNA extractor Trizol. The PrimeScript RT Master Mix reverse transcription kit was used to generate complementary DNA (cDNA) as a qRT-PCR template. Relative qRT-PCR was used to measure the PEDV nucleocapsid protein (N) RNA content to represent the viral RNA content of infected cells. Viral RNA was normalized with actin β-actin mRNA and quantified by 2 -△△CT Methods: Relative quantification was performed; primers and reaction systems are shown in Tables 1 and 2; the staining method fluorescence quantitative premix was AceQ Universal SYBR qPCR master Mix; the reaction program used the Fast program provided with the ABI fluorescence quantitative PCR instrument.

[0046] Table 1. qRT-PCR primers are as follows

[0047]

[0048] Table 2. Reaction system and procedure

[0049]

[0050] The experiment was performed three times independently with three replicates each time. The experimental data are expressed as the group mean and standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software with unpaired, two-tailed Student t-test. ***p<0.001 indicated extremely significant statistical differences.

[0051] The results are as follows Figure 7 and Figure 8As shown, compared with the DMSO group, the relative expression levels in the baicalein treatment groups at 1 μmol / L to 2 μmol / L were higher than 50%, and when the concentration was higher than 4 μmol / L, the relative expression levels approached 0, indicating that baicalein above 4 μmol / L could significantly reduce the intracellular PEDV particle content; the relative expression level in the gemcitabine treatment group at 1 μmol / L was higher than 50%, the relative expression levels in the treatment groups at 2 μmol / L to 4 μmol / L were lower than 50%, and the relative expression level in the 6 μmol / L treatment group approached 0, indicating that gemcitabine inhibited the relative expression of the PEDV N gene in a dose-dependent manner.

[0052] Example 4: Effect of Baicalein / Gemcitabine on the Viral Titer of PEDV-Infected Vero Cells

[0053] To verify whether baicalein / gemcitabine can reduce the viral titer of PEDV-infected Vero cells, Vero cells were cultured according to Example 3, different concentrations of baicalein / gemcitabine drugs were diluted (the concentration and control group settings were the same as in Example 2), and 0.1 MOI of the virus was inoculated. After collecting the cells and the supernatant, the uninfected Vero cells were diluted to 2.5×105 cells / mL and plated in a 96-well cell culture plate, 100 μL of DMEM medium was added to each well, and it was cultured overnight in an incubator at 37°C and 5% CO2; the cell supernatant infected for 16 h above was serially diluted 10-fold, and inoculated into the Vero cells plated in the 96-well cell culture plate, 100 μL was inoculated into each well, and 8 replicates were made for each dilution. In addition, the supernatant of PEDV-infected Vero cells without drug treatment was used as the control group; the 96-well cell culture plate was placed in an incubator at 37°C and 5% CO2 for culture; the growth state of the cells was observed once every 24 h, and the number of cytopathic wells was recorded. Observation was carried out for 2 d until the number of cytopathic wells no longer increased; the TCID50 of the virus was calculated by the Reed-Muench method; the experimental data were expressed as the group mean and standard deviation (SD), and statistical analysis was performed using the unpaired, two-tailed Student t-test of GraphPad Prism 8.0 software; ***p < 0.01 indicated extremely significant statistical differences.

[0054] The results are as Figure 9 and Figure 10 shown. Compared with the DMSO group, as the concentration of the drug treatment group increased, the viral titer gradually decreased. The TCID50 of the 10 μmol / L baicalein treatment group decreased by 3.8 log10; compared with the DMSO group, the viral titer of the 10 μmol / L gemcitabine treatment group decreased by 4 log10. The above results indicate that the anti-PEDV activity of baicalein / gemcitabine is concentration-dependent. Through calculation and analysis, the EC 50 of baicalein was 5.01 μmol / L, and the EC 50 of gemcitabine was 3.14 μmol / L.

[0055] Example 5: Effects of Baicalein / Gemcitabine on the Proliferation Cycle of Vero Cells Infected with PEDV

[0056] To further confirm the inhibition of Baicalein / Gemcitabine on the level of PEDV-infected Vero cells, drug treatment groups of 5 μmol / L and 10 μmol / L were set for both compounds. Vero cells were cultured and infected with PEDV particles according to Example 1, and cell fluid and supernatant were collected. The virus content in the samples was detected by TCID50 experiment. The test methods at different proliferation stages are as follows:

[0057] (1) Inactivation stage: Different concentrations of drugs were mixed with 0.1 MOI PEDV virus and incubated in a 37°C incubator for 2 h. The above mixture was inoculated onto cells and cultured at 37°C for 1 h. The mixture was discarded, and the plate was washed 3 times with serum-free DMEM. Then, it was replaced with virus growth medium and cultured until 12 h later for sample collection.

[0058] (2) Adsorption stage: Vero cells were pre-cooled at 4°C for 30 min, inoculated with 0.1 MOI PEDV + a mixture containing / without drugs, and incubated at 4°C for 1 h. The plate was washed 3 times with serum-free DMEM, then replaced with virus growth medium and placed in an incubator for 12 h later for sample collection.

[0059] (3) Internalization stage: Vero cells were pre-cooled at 4°C for 30 min, inoculated with 0.1 MOI PEDV and incubated at 4°C for 1 h. The plate was washed 3 times with serum-free DMEM, then medium containing / without drugs was added, and the plate was washed after culturing at 37°C for 2 h. Then, it was replaced with virus growth medium and placed in an incubator for 12 h later for sample collection.

[0060] (4) Replication stage: 0.1 MOI PEDV infected Vero cells and were cultured at 37°C for 2 h. After washing three times with PBS, it was replaced with medium containing / without drugs, and the plate was washed after standing at 37°C for 2 h. Then, it was replaced with virus growth medium and placed in an incubator for 12 h later for sample collection.

[0061] (5) Release stage: 0.1 MOI PEDV infected Vero cells and were incubated at 37°C for 12 h. After washing the plate 3 times, medium containing / without the compound was added, and supernatant samples were collected every 15 min / 30 min / 45 min / 60 min after virus inoculation for sample collection.

[0062] The results are as Figure 11 and Figure 12As shown in the figure, compared with the DMSO group, the virus titer decreased with the increase of baicalein concentration. The virus titer in the 10 μmol / L baicalein treatment group decreased by 2 log10, indicating that baicalein has the effect of directly inactivating PEDV; the results of the adsorption stage showed that compared with the DMSO group, the virus titer increased slightly with the increase of baicalein concentration; the internalization stage showed that compared with the DMSO group, the virus titer in the 5 μmol / L baicalein treatment group was similar, and the virus titer in the 10 μmol / L treatment group decreased; in the replication stage, the virus titers of the baicalein treatment groups with different concentrations were similar to those of the DMSO group, indicating that baicalein has a slight effect on the replication stage of PEDV; the release stage showed that there were no significant differences in the virus titers between the baicalein treatments with different concentrations and different time periods and the DMSO group, indicating that baicalein does not affect the release stage of PEDV. The above results show that baicalein mainly acts on the early entry stage of the PEDV proliferation cycle.

[0063] The virus titers of the gemcitabine treatment groups with different concentrations were almost the same as those of the DMSO group in the adsorption, internalization, and release stages; the results of the replication stage showed that the virus titer decreased significantly with the increase of gemcitabine concentration. The virus titer in the 10 μmol / L gemcitabine treatment group decreased by 2.4 log10 compared with the DMSO group. The above results show that gemcitabine mainly affects the replication stage of the PEDV proliferation cycle.

[0064] Example 6: Effect of Baicalein Combined with Gemcitabine on the Virus Titer of PEDV-Infected Vero Cells

[0065] Based on the mechanism that the two compounds act on different stages of PEDV proliferation, the two were used in combination to verify whether baicalein combined with gemcitabine could have a synergistic effect. Vero cells were cultured according to Example 1. First, taking the EC 50 values of the two compounds as the gradient centers, concentration gradients were set at 0.125 / 0.25 / 0.5 times: the gemcitabine concentrations were: 0.5 μmol / L, 1 μmol / L, 2 μmol / L; the baicalein concentrations were: 0.75 μmol / L, 1.5 μmol / L, 3 μmol / L for cross-combination to explore the anti-PEDV activity. The two drugs were used in cross-combination at each concentration and co-incubated with 0.1 MOI PEDV to explore the effect of combined drug use on PEDV activity. After co-incubating with the virus at 37 °C for 2 h, it was inoculated into cell wells, and samples were collected after 16 h for TCID 50 assay.

[0066] After collecting the cells and supernatants, the uninfected Vero cells were diluted to 2.5×10 5Seed 96-well cell culture plates with cells at a density of [number of cells] cells / mL. Add 100 μL of DMEM medium to each well and incubate overnight in an incubator at 37 °C and 5% CO₂. Take the cell supernatant of the cells infected for 16 h as described above and dilute it 10-fold. Inoculate Vero cells seeded in 96-well cell culture plates with 100 μL per well. Make 8 replicates for each dilution. Additionally, use the supernatant of Vero cells infected with PEDV without drug treatment as the control group. Place the 96-well cell culture plates in an incubator at 37 °C and 5% CO₂ for culture. Observe the growth status of the cells once every 24 h and record the number of wells with cytopathic effects. Observe for 2 days until the number of wells with cytopathic effects no longer increases. Calculate the TCID of the virus using the Reed-Muench method. 50 The experimental data are expressed as the group mean and standard deviation (SD). Statistical analysis is performed using unpaired, two-tailed Student's t-test with GraphPad Prism 8.0 software; ***p < 0.01 indicates a highly significant statistical difference.

[0067] The results are as Figures 13 to 15 shown. Compared with each group treated with a single drug alone, the virus titers in the combined drug treatment groups decreased significantly. Compared with the DMSO group, the virus titer decreased by 2.5 log10 in the group treated with 0.75 μmol / L baicalein and 0.5 μmol / L gemcitabine. Using GraphPad Prism 8 for fitting calculation, the inhibition rate of PEDV in the group treated with 0.75 μmol / L baicalein combined with 2 μmol / L gemcitabine reached 54%. Further, use Synergy finder software to calculate the CI value of the combined drug. The CI < 1 for the combined drug at each concentration, indicating that gemcitabine combined with baicalein has a synergistic effect. The inhibition rate and ZIP synergy index of the combined drug are obtained, as shown in Table 3.

[0068] Table 3. Calculation results of the CI value of the combined drug

[0069]

[0070] Note: In the table, CI > 1 indicates antagonistic effect, CI = 1 indicates additive effect, and CI < 1 indicates synergistic effect.

[0071] Example 7: Verification of the effect of baicalein combined with gemcitabine on inhibiting PEDV infection of Vero cells

[0072] Cross-configure combined drugs with different concentrations according to Example 6 and conduct antiviral tests. After collecting samples, discard the cell supernatant. Fix the cells with 4% paraformaldehyde, incubate with PEDV N antibody at room temperature for 2 h, incubate with fluorescent secondary antibody in the dark for 1 h, incubate with DAPI for 15 min, and observe the fluorescence results using an inverted microscope.

[0073] The results are as Figure 16As shown, the green fluorescence expression in the combined drug treatment group gradually weakened and disappeared with the increase of the combined concentration. The above results all confirmed that the combined drug can reduce the required concentration of the two drugs and has significant anti-PEDV activity.

[0074] Example 8: Identification of the cytotoxicity of baicalein combined with gemcitabine on Vero cells

[0075] Cross-configure the combined drugs with different concentrations according to Example 6. For the well-growing Vero cells, seed them in a 96-well cell culture plate at a density of 2.5×10 5 cells / mL. Add 100 μL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin to each well, and culture in a 37°C, 5% CO2 incubator for 24 h; remove the original cell culture medium, replace it with DMEM medium containing the combined drugs, incubate at 37°C for 24 h, then add 10 μL of CCK-8 reagent and culture in a 37°C, 5% CO2 incubator for 2 h; perform cytotoxicity according to the operation instructions.

[0076] The results are as Figure 17 shown. The viable cells in the combined drug group are higher than 80%, indicating that the combined drug concentration does not cause toxic killing to Vero cells.

[0077] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. In addition, it should be understood that although this specification is described according to the embodiments, it does not only contain one technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Use of baicalin combined with gemcitabine in the preparation of an anti-porcine epidemic diarrhea virus drug, wherein the molar ratio of baicalin to gemcitabine is 0.75-3:0.5-2.

2. The use according to claim 1, characterized in that: The molar ratio of baicalein to gemcitabine is 0.75:0.5.

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