Application of illicium henryi or illicium henryi extract in preparation of medicine for resisting porcine epidemic diarrhea virus

Abstract

The invention relates to an application of illicium henryi or an extract of the illicium henryi in preparation of a medicine for resisting porcine epidemic diarrhea viruses. The illicium henryi is a Magnoliaceae plant, namely, Illicium lanceolatum A.C.Smith. The invention further relates to a preparation method of the illicium henryi and the extract of the illicium henryi. Researches show that the illicium henryi extract can effectively and directly kill viruses, block the viruses from entering cells, inhibit the viruses from being copied in the cells and play a role in resisting the porcine epidemic diarrhea viruses, and can be applied to preparation of the medicine for resisting the porcine epidemic diarrhea viruses.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of red anise or its extract in the preparation of drugs against swine epidemic diarrhea virus. The red anise is the dried root bark or stem bark of Illicium lanceolatum ACSmith, a plant of the Magnoliaceae family. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Infected pigs mainly exhibit clinical symptoms such as diarrhea, vomiting, anorexia, dehydration, and weight loss. Adult pigs often present with asymptomatic infection, while newborn piglets are frequently severely infected, with a mortality rate that can reach 100%, causing enormous economic losses to the global pig industry. Although several PED vaccines are available, frequent viral mutations result in poor protection for newborn piglets, and even immunized herds can still become infected. Furthermore, there are currently no specific drugs for PED in clinical practice, and synthetic chemical antiviral drugs have significant side effects, are prone to drug resistance, and have very limited clinical efficacy. Since 2005, my country has completely banned the use of antiviral drugs such as amantadine, rimantadine, ribavirin, and acyclovir in foodborne animals. Therefore, the development of drugs against PEDV is of great significance and practical value for the prevention and control of PED.

[0003] Red anise is the dried root bark or stem bark of *Illicium lanceolatum* ACSmith, a plant in the Magnoliaceae family. It has a pungent taste and warm properties; it is used to dispel wind and unblock meridians, relax muscles and promote blood circulation, and relieve pain. It can treat injuries from falls and blows, lumbar muscle strain, rheumatic pain, carbuncles, and boils. The root bark or stem bark is extracted, purified, filtered, and sterilized using an alcohol extraction process to obtain an injection solution, which has been widely used clinically to treat chronic low back and leg pain, lumbar muscle strain, lumbar disc herniation, rheumatic joint diseases, sciatica, and frozen shoulder, and has a high safety profile. Studies have reported on the antiviral effects of red anise against pseudorabies virus and porcine reproductive and respiratory syndrome virus. However, to date, there are no reports on the antiviral effects of red anise against porcine epidemic diarrhea virus.

[0004] The present invention demonstrates that red fennel or its extracts can effectively and directly kill viruses in vitro, block viral entry into cells, and inhibit viral replication in cells, thus exerting an antiviral effect against swine epidemic diarrhea virus. Summary of the Invention

[0005] The primary objective of this invention is to provide a novel drug with anti-swine epidemic diarrhea virus activity, specifically, the drug comprising red anise extract.

[0006] A second object of the present invention is to provide a pharmaceutical composition for treating swine epidemic diarrhea virus.

[0007] A third object of the present invention is to provide the use of the above-mentioned red anise or the above-mentioned red anise extract or the composition in the preparation of a drug for the prevention and / or treatment of porcine epidemic diarrhea.

[0008] The red anise with anti-swine epidemic diarrhea virus activity in this invention is Illicium lanceolatum ACSmith, a plant of the genus Illicium in the family Magnoliaceae.

[0009] The red anise in this invention includes fresh or dried red anise (I. lanceolatum ACSmith); the medicinal parts can be the whole plant, root, root bark, stem, stem bark or any combination thereof; the preferred medicinal parts are root, root bark, stem, stem bark or any combination thereof; the more preferred medicinal parts are root bark or stem bark; the most preferred medicinal part is root bark.

[0010] The present invention provides a pharmaceutical composition for combating swine epidemic diarrhea virus containing a preventive or therapeutically effective amount of red anise or its extract and a pharmaceutically acceptable carrier.

[0011] The red anise or its extract or pharmaceutical composition of the present invention can be used for the prevention and / or treatment of porcine epidemic diarrhea, wherein the red anise or its extract has any of the following effects: 1) Red anise or its extracts inhibit the proliferation and replication of porcine epidemic diarrhea virus in cells; 2) Red anise or its extracts inhibit the infection of cells by porcine epidemic diarrhea virus; 3) Red anise or its extracts can kill porcine epidemic diarrhea virus; 4) Red anise or its extracts can block the adsorption of porcine epidemic diarrhea virus to cells.

[0012] The pharmaceutically acceptable carriers mentioned above refer to excipients and carriers in the pharmaceutical field.

[0013] The pharmaceutical compositions of the present invention are administered to subjects requiring such prevention or treatment via oral, nasal, rectal, parenteral, or transdermal routes.

[0014] Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional pharmaceutical manufacturing methods. For example, the active ingredient can be mixed with one or more carriers and then formulated into the desired dosage form.

[0015] The red anise extract of this invention can effectively and directly kill viruses, block viral entry into cells, and inhibit viral replication in cells, thus exerting an antiviral effect against porcine epidemic diarrhea virus (PEDV). It can be used in the preparation of anti-PEDV drugs. The antiviral effect of red anise extract against PEDV is significantly superior to that of ribavirin (RBV), showing promising development and application prospects. Attached Figure Description

[0016] Figure 1 The effect of red anise extract on Vero cell proliferation. Where A: 2h, B: 6h; *** indicates a significant difference at the 0.001 level compared to 0 μg / mL.

[0017] Figure 2 The study investigated the antiviral activity of red anise extract against porcine epidemic diarrhea virus (PEDV), where *** indicates a significant difference at the 0.001 level compared to the PEDV group.

[0018] Figure 3 This study investigated the effect of red anise extract on the fluorescence intensity of Vero cells infected with porcine epidemic diarrhea virus carrying the green fluorescent protein reporter gene.

[0019] Figure 4 The study investigated the killing effect of red anise extract on porcine epidemic diarrhea virus (PEDV). *** indicates a significant difference at the 0.001 level compared to the PEDV group.

[0020] Figure 5 The study investigated the blocking effect of red anise extract on the adsorption of porcine epidemic diarrhea virus (PEDV) onto Vero cells. *** indicates a significant difference at the 0.001 level compared to the PEDV group.

[0021] Figure 6 The study investigated the inhibitory effect of red anise extract on porcine epidemic diarrhea virus replication. *** indicates a significant difference at the 0.001 level compared to the PEDV group. Detailed Implementation

[0022] The invention is further illustrated below with examples and accompanying drawings, but is not intended to limit its scope.

[0023] Example 1: Preparation of Red Anise Extract

[0024] Red anise medicinal material was taken, and 10 times the amount of 75% ethanol was added. The mixture was refluxed and extracted twice, 2 hours each time. The extracts were filtered, and the filtrates were combined. The ethanol was recovered under reduced pressure until there was no alcohol odor. The extract was then concentrated and freeze-dried to obtain red anise extract (HHXE) with a yield of 21.50%.

[0025] Example 2: Preparation of Red Fennel Injection

[0026] Take 50g of red anise root bark, add 250ml of 75% ethanol, soak for 7-10 days, filter, wash the residue with a small amount of 75% ethanol, filter again, combine the filtrates, recover the ethanol until no alcohol odor remains, let stand, cool, take the supernatant, add 400ml of water for injection, stir well, adjust the pH to about 7.0 with 10% sodium hydroxide solution, refrigerate overnight, filter, add 20ml of polysorbate 80 to the filtrate, stir well, add an appropriate amount of water for injection, adjust the pH to about 9.7 with 10% sodium hydroxide solution, add water for injection to make 1000ml, stir well, filter, fill, sterilize, and obtain red anise injection (HHXI). Each ml contains 50mg of raw red anise.

[0027] Example 3: Effects of Red Fennel Extract on Vero Cell Proliferation

[0028] 0.75×10 5 African green monkey kidney Vero cell suspension (cells / mL) was seeded at 100 μL per well in 96-well plates and incubated at 37°C, 5% CO2 for 24 h until a monolayer formed. The culture medium was discarded, and 100 μL of DMEM culture medium or different concentrations of red anise extract (HHXE) diluted to each well (equivalent to 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mg / mL of red anise medicinal material) was added. The plates were then incubated at 37°C, 5% CO2 for 2 h and 6 h, respectively. The supernatant was discarded, and 100 μL of porcine epidemic diarrhea virus (PEDV) maintenance medium (DMEM culture medium containing 5 μg / mL trypsin) was added to each well. The plates were then incubated at 37°C, 5% CO2 for 46 h and 42 h, respectively (48 h from the start of drug action). Four hours before the end of the culture period, add 50 μL of 2 mg / mL MTT solution to each well, shake well, and allow the crystals to dissolve completely. Measure the OD value at 490 nm using a microplate reader.

[0029] The results are as follows Figure 1 As shown, the concentration of red anise extract (HHXE) greater than 1.2 mg crude drug / mL significantly inhibited the proliferation of Vero cells in the kidneys of African green monkeys (P<0.001), and its maximum safe concentration (MNTC) for Vero cells was 1.2 mg crude drug / mL.

[0030] Example 4: Antiviral effect of red anise extract against porcine epidemic diarrhea virus

[0031] The concentration was 0.75 × 10⁻⁶. 5Vero cell suspension at 100 μL / well was seeded into 96-well plates and incubated at 37°C and 5% CO2 for 24 h until a monolayer formed. The culture medium was discarded, and 100 μL of DMEM or different concentrations of HHXE (equivalent to 0.2, 0.4, and 0.8 mg / mL of anise extract) was added to each well. The plates were then incubated at 37°C and 5% CO2 for 2 h. The supernatant was discarded, and each well was first added to DMEM containing 10 μg / mL trypsin or 200 TCID50. 50 Add 50 μL of porcine epidemic diarrhea virus (PEDV) dilution to DMEM culture medium or 50 μL of different concentrations of red anise extract (HHXE) dilution (equivalent to 0.4, 0.8, and 1.6 mg / mL of red anise raw material). Incubate at 37°C in a 5% CO2 incubator, shaking well every 30 minutes. After 2 hours of incubation, discard the virus-drug mixture in each well. Add 100 μL of DMEM culture medium or different concentrations of red anise extract dilution (equivalent to 0.2, 0.4, and 0.8 mg / mL of red anise raw material) to each well and incubate at 37°C in a 5% CO2 incubator for 2 hours. Discard the supernatant and add 100 μL of PEDV-specific maintenance medium (DMEM culture medium containing 5 μg / mL trypsin) to each well. Incubate at 37°C in a 5% CO2 incubator for 42 hours. Four hours before the end of the culture, 50 μL of 2 mg / mL MTT solution was added to each well, and the mixture was shaken until the crystals were completely dissolved. The OD value was measured at 490 nm using a microplate reader. The inhibition rate of porcine epidemic diarrhea virus (PEDV) proliferation in Vero cells was calculated using the following formula. A ribavirin (RBV) group was also set up as a positive control.

[0032] Inhibition rate (%) = (OD value of experimental group - OD value of virus control group) / (OD value of cell control group - OD value of virus control group) × 100%.

[0033] The results are as follows Figure 2 As shown, full-course administration of red anise extract (HHXE) significantly inhibited the proliferation of porcine epidemic diarrhea virus (PEDV) in Vero cells in a concentration-dependent manner. Full-course administration of red anise extract at concentrations of 0.2, 0.4, and 0.8 mg crude drug / mL resulted in inhibition rates of 84.17%, 97.59%, and 98.68% against PEDV proliferation in Vero cells, respectively, significantly superior to the positive control drug ribavirin (RBV), which showed an inhibition rate of only 45.38%.

[0034] Example 5: Effect of red anise extract on fluorescence intensity of Vero cells infected with porcine epidemic diarrhea virus carrying green fluorescent protein reporter gene

[0035] The strain used was porcine epidemic diarrhea virus (PEDV-GFP) carrying the green fluorescent protein reporter gene. Vero cells cultured adherently for 24 hours were used in 24-well plates. Cell control, virus control, ribavirin (RBV) control, and red anise extract (HHXE) experimental groups were set up, and cells were treated with drugs and viruses according to the method in Example 4. The culture medium was discarded, and 300 μL of 1× Hoechst 33342 viable cell staining solution was added to each well. The cells were incubated in an incubator for 10 min, the culture medium was discarded, phosphate buffer (pH 7.4) was added, and the cells were observed and photographed under a fluorescence microscope.

[0036] The results are as follows Figure 3 As shown, red anise extract (HHXE) can reduce the green fluorescence intensity of porcine epidemic diarrhea virus (PEDV-GFP) infected Vero cells in a concentration-dependent manner. The green fluorescence intensity of PEDV-GFP infected Vero cells treated with red anise extract is significantly lower than that of the positive control drug ribavirin (RBV). In particular, almost no green fluorescence can be observed in PEDV-GFP infected Vero cells treated with red anise extract at a concentration of 0.4 mg crude drug / mL, further confirming that red anise extract (HHXE) has an anti-porcine epidemic diarrhea virus (PEDV) effect.

[0037] Example 6: Killing effect of red anise extract on porcine epidemic diarrhea virus

[0038] Mix DMEM culture medium or diluted red anise extract (HHXE) at different concentrations (equivalent to 0.2, 0.4, and 0.8 mg / mL of red anise raw material) with 200 TCID2. 50 Equal volumes of porcine epidemic diarrhea virus (PEDV) dilution were mixed and incubated at 37°C in a 5% CO2 incubator for 2 hours. 100 μL of the mixture was added to each well of a 96-well plate containing adherent Vero cells cultured for 24 hours to form a monolayer, with four replicates. A blank control, normal cell control, virus control, and ribavirin (RBV) control group were also included. The plates were incubated at 37°C in a 5% CO2 incubator, with shaking every 30 minutes. After 2 hours of incubation, the virus-drug mixture was discarded, and 100 μL of PEDV-specific maintenance medium (DMEM medium containing 5 μg / mL trypsin) was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 70 hours, with daily observation of cytopathic effects (CPE). Four hours before the end of the culture period, 50 μL of 2 mg / mL MTT solution was added to each well, and the plates were shaken until the crystals were completely dissolved. The OD value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the kill rate against porcine epidemic diarrhea virus was calculated using the following formula.

[0039] Killing rate (%) = (OD value of experimental group – OD value of virus control group) / (OD value of cell control group – OD value of virus control group) × 100%.

[0040] The results are as follows Figure 4 As shown, red anise extract (HHXE) exhibited significant bactericidal activity against porcine epidemic diarrhea virus (PEDV) in a concentration-dependent manner. Concentrations of 0.1, 0.2, and 0.4 mg crude drug / mL of red anise extract resulted in PEDV kill rates of 45.25%, 83.56%, and 94.85%, respectively, significantly superior to the positive control drug ribavirin (RBV), which had a kill rate of only 16.55%.

[0041] Example 7: Blocking effect of red anise extract on porcine epidemic diarrhea virus adsorption on Vero cells

[0042] The concentration was 0.75 × 10⁻⁶. 5 African green monkey kidney Vero cell suspension (cells / mL) was seeded at 100 μL per well in 96-well plates and incubated at 37°C, 5% CO2 for 24 h until a monolayer formed. The culture medium was discarded, and 100 μL of DMEM culture medium or different concentrations of red anise extract (HHXE) dilution (equivalent to 0.1, 0.2, and 0.4 mg / mL of red anise raw material) was added to each well. The plates were then incubated at 37°C, 5% CO2 for 2 h. The supernatant was discarded, and DMEM culture medium containing 5 μg / mL trypsin or 100 TCID50 was added to each well. 50 100 μL of porcine epidemic diarrhea virus (PEDV) dilution was incubated in a 37°C, 5% CO2 incubator, with shaking every 30 min. After 2 h of incubation, the supernatant was discarded, and 100 μL of PEDV-specific maintenance medium (DMEM medium containing 5 μg / mL trypsin) was added to each well. The wells were then incubated at 37°C, 5% CO2 for 44 h. Four h before the end of the incubation period, 50 μL of 2 mg / mL MTT solution was added to each well, and the mixture was shaken until the crystals were completely dissolved. A ribavirin (RBV) group was also included as a positive control. OD values ​​were measured at 490 nm using a microplate reader. The blocking rate of PEDV adsorption on Vero cells was calculated using the following formula.

[0043] Blocking rate (%) = (OD value of experimental group – OD value of virus control group) / (OD value of cell control group – OD value of virus control group) × 100%.

[0044] The results are as follows Figure 5As shown, red anise extract (HHXE) significantly blocked porcine epidemic diarrhea virus (PEDV) from entering Vero cells in a concentration-dependent manner. The PEDV blocking rates of red anise extract at concentrations of 0.1, 0.2, and 0.4 mg crude drug / mL were 34.97%, 58.65%, and 92.43%, respectively, significantly superior to the positive control drug ribavirin (RBV), which had a kill rate of only 21.49%.

[0045] Example 8: Inhibitory effect of red anise extract on porcine epidemic diarrhea virus replication

[0046] The concentration was 0.75 × 10⁻⁶. 5 African green monkey kidney Vero cell suspension (cells / mL) was seeded at 100 μL per well in 96-well plates and incubated at 37°C in a 5% CO2 cell culture incubator for 24 h until a monolayer formed. The culture medium was discarded, and each well was first filled with DMEM culture medium containing 5 μg / mL trypsin or 100 TCID50. 50 100 μL of each diluted porcine epidemic diarrhea virus (PEDV) solution was incubated in a 37°C, 5% CO2 incubator, and shaken every 30 min. After 2 h of incubation, the virus solution in each well was discarded. 100 μL of DMEM culture medium or diluted red anise extract at different concentrations (equivalent to 0.1, 0.2, and 0.4 mg / mL of red anise medicinal material) was added to each well, and the wells were incubated in a 37°C, 5% CO2 incubator for 2 h. The supernatant was discarded, and 100 μL of PEDV-specific maintenance medium (DMEM culture medium containing 5 μg / mL trypsin) was added to each well, and the wells were incubated in a 37°C, 5% CO2 incubator for 46 h. Four h before the end of the incubation period, 50 μL of 2 mg / mL MTT solution was added to each well, and the wells were shaken until the crystals were completely dissolved. A ribavirin (RBV) group was also included as a positive control. The OD value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate against porcine epidemic diarrhea virus replication was calculated using the following formula.

[0047] Inhibition rate (%) = (OD value of experimental group – OD value of virus control group) / (OD value of cell control group – OD value of virus control group) × 100%.

[0048] The results are as follows Figure 6 As shown, red anise extract (HHXE) significantly inhibited porcine epidemic diarrhea virus (PEDV) replication in Vero cells in a concentration-dependent manner. Specifically, the inhibition rate of red anise extract (HHXE) at a concentration of 0.4 mg crude drug / mL was 39.76%, which was not significantly different from the positive control drug ribavirin (RBV), whose inhibition rate was 38.99%.

[0049] In summary, the red anise extract of this invention can effectively and directly kill viruses, block viral entry into cells, and inhibit viral replication in cells, thus exerting an antiviral effect against porcine epidemic diarrhea virus (PEDV). It can be used in the preparation of anti-PEDV drugs. The antiviral effect of red anise extract against PEDV is significantly superior to that of ribavirin (RBV), demonstrating promising development and application prospects.

Claims

1. The application of red anise or its extract in the preparation of drugs against porcine epidemic diarrhea virus, characterized in that, The red anise mentioned is Illicium lanceolatum ACSmith, a plant belonging to the Magnoliaceae family.

2. The use of red anise or its extract according to claim 1 in the preparation of drugs against porcine epidemic diarrhea virus, characterized in that, The drug uses red anise or its extract as its active ingredient.

3. The use of red anise or its extract according to claim 1 in the preparation of drugs against porcine epidemic diarrhea virus, characterized in that, The red anise or its extract has any of the following effects: 1) Red anise or its extracts inhibit the proliferation and replication of porcine epidemic diarrhea virus in cells; 2) Red anise or its extracts inhibit the infection of cells by porcine epidemic diarrhea virus; 3) Red anise or its extracts can kill porcine epidemic diarrhea virus; 4) Red anise or its extracts can block the adsorption of porcine epidemic diarrhea virus to cells.

4. A drug for treating swine epidemic diarrhea virus, characterized in that, The active ingredient is red anise or its extract.

5. The drug as described in claim 4, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

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