CN108524476B - Application of xanthohumol in preparation of medicine for resisting porcine epidemic diarrhea virus - Google Patents

Abstract

The invention discloses an application of xanthohumol in preparation of a medicament for resisting porcine epidemic diarrhea virus, wherein the mass percentage content of the xanthohumol is not less than 99.75%, the application dose of the xanthohumol is 1-10 mu M/L, and the xanthohumol has a very obvious effect of resisting the porcine epidemic diarrhea virus, is safe, has few toxic and side effects, and is low in medicament residue and free of pollution.

Description

Application of xanthohumol in preparation of medicine for resisting porcine epidemic diarrhea virus

Technical Field

The invention belongs to the field of antiviral application, and particularly relates to application of xanthohumol in preparation of a medicament for resisting porcine epidemic diarrhea viruses.

Background

Porcine epidemic diarrhea is a disease caused by Porcine Epidemic Diarrhea Virus (PEDV) and is clinically characterized by vomiting, severe diarrhea and dehydration, which are frequently epidemic in winter and can infect pigs at all ages. The suckling piglets are most susceptible, the morbidity can reach 100 percent, the fatality rate can reach 80 to 100 percent, replacement pigs, sows or fattening pigs are in transient diarrhea, and the morbidity is low. PEDV appeared for the first time in europe in the seventies of the last century, however, since 2010, the outbreak of the variant virus strain of PEDV caused huge economic losses to the pig industry in asian countries such as china, japan, korea, thailand, philippines, etc., and was also prevalent in countries such as the united states, germany, and vitta, respectively, in 2013, the prevalence of the variant virus strain of PEDV had severely affected the global pig industry, particularly suckling piglets, and the existing prevention and control measures have not been able to cope with new flow state.

The porcine epidemic diarrhea virus is often mixed with transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) to cause clinical symptoms such as vomiting, severe diarrhea and dehydration, and the PEDV positive rate separated from each pig farm is the highest from the pig farm viral diarrhea epidemiological survey result in the middle part of the year 2012 plus 2017. The porcine epidemic virus is a member of Coronaviridae (Coronaviridae) Coronaviridae (Coronaviridae) of Nidovirales (Nidovirales), is a single-stranded positive-strand RNA virus, is spherical in appearance, has the diameter of about 95-190nm, is wrapped by an envelope outside a virus particle, has 12-24nm fiber protrusions on the surface of the envelope, has spherical fiber protrusions at the tail ends, and is orderly arranged into a crown shape. PEDV virions are less sensitive to heat, and survive stably at pH5.0-9.0 at 4 ℃ and pH6.5-7.5 at 37 ℃, but are inactivated at 60 ℃ for 30 min. Furthermore, PEDV is sensitive to lipid soluble liquids and the virion does not possess hemagglutination properties.

The PEDV viral genome is 28kb in length, comprises a 5 'cap structure and a 3' adenosine tail structure, comprises at least 7 Open Reading Frames (ORFs), encodes 3 non-structural proteins (replicase 1a, 1b and ORF3) and 4 structural proteins: 150-220kDa glycosylated spike (S) protein, 20-30kDa membrane (M) protein, 7kDa envelope (E) protein and 58kDa nucleocapsid (N) protein. Research shows that viruses invade cells through protein receptor aminopeptidase N-acetylcholine neuraminic acid, the receptors are also present in human, monkey, bat and other species, and PEDV has potential risk of cross-species infection, so that research on anti-PEDV drugs has an important role.

Disclosure of Invention

Xanthohumol (Xanthohumol) is an isoprene flavonoid compound emutextracted from hops, hops (Humulus lupulus L) also known as hop (common English name), hops and hops belong to perennial herb plants of Humulus in Moraceae and are one of main raw materials for brewing beer, the Xanthohumol is secreted by hop lupin glands and is found only in hops at present and is one of main flavonoid substances in hops.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect of the invention, use of xanthohumol in the preparation of a medicament for the treatment of porcine epidemic diarrhea virus.

In a second aspect of the invention, a medicament for resisting porcine epidemic diarrhea virus comprises xanthohumol and pharmaceutically acceptable auxiliary materials or auxiliary components.

Preferably, in the medicine for resisting porcine epidemic diarrhea virus, the content of the xanthohumol in percentage by mass is not less than 99.75%, and the application dose is 1-10 μ M/L, namely 354.4 μ g/L-3.544 mg/L.

In some preferred embodiments, the anti-porcine epidemic diarrhea virus drug can be a tablet, a powder, a granule, a capsule, an oral liquid, an injection or a sustained release agent.

Compared with the prior art, the invention has the beneficial effects that:

firstly, the xanthohumol has very obvious effect of resisting porcine epidemic diarrhea virus; experiments prove that the xanthohumol shows obvious antiviral activity in cell experiments, and can show extremely strong anti-porcine epidemic diarrhea virus (MOI ═ 0.01) infection effect at the concentration of 10 mu M.

Secondly, the xanthohumol is safe and has little toxic and side effects when being used for resisting porcine epidemic diarrhea virus; xanthohumol is an extracted component in Chinese herbal medicines, is different from hormones, antibiotics, chemical synthetic medicines and the like, and has no obvious toxic or side effect on organisms.

Thirdly, the xanthohumol used for resisting the porcine epidemic diarrhea virus has low drug residue and no pollution; the xanthohumol belongs to an organic molecular compound, is easy to be absorbed by animal bodies, has high biological metabolism rate and is discharged without pollution.

Drawings

FIG. 1 is a MTT method for detecting toxicity of xanthohumol on Vero-E6 cells; treating Vero-E6 cells with 1-10 μ M xanthohumol for 72h, staining for 4h by using MTT, and detecting cell activity by measuring cell absorbance at 490 nm;

FIG. 2 is a Western Blot to determine the activity of xanthohumol in inhibiting porcine epidemic diarrhea virus infection on Vero-E6 cells; 1-10 mu M xanthohumol is used for pretreating Vero-E6 cells for 2h and then infecting porcine epidemic diarrhea viruses, the xanthohumol exists all the time, and the cells are collected after 24h and then Western Blot is carried out to determine the protein content of PEDV in the cells;

FIG. 3 shows inhibition of PEDV infection by xanthohumol on Vero-E6 cells as determined by fluorescent quantitative PCR; pretreating Vero-E6 cells with 1-10 mu M of xanthohumol for 2h, then infecting porcine epidemic diarrhea virus, then allowing the xanthohumol to exist all the time, collecting the cells after 24h, extracting total RNA, and performing fluorescence quantitative PCR determination;

FIG. 4 is a plaque formation assay to determine that xanthohumol inhibits PEDV infection on Vero-E6 cells; Vero-E6 cells were pretreated with 10 μ M xanthohumol for 2h before infecting porcine epidemic diarrhea virus, after which xanthohumol was present all the time, and after 24h the supernatant was collected and assayed for virus titer using plaque formation assay.

Detailed Description

The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following embodiments.

Test materials

The porcine epidemic diarrhea virus H L JBY strain is owned by the inventor laboratory;

Vero-E6 cells (African green monkey kidney cells) were given to the university of agriculture immune research institute of Nanjing;

the MTT assay kit was purchased from Solebao corporation;

real-time quantitative PCR (real time PCR) AceQ

The Green Master Mix kit was purchased from nozak;

primers were ordered from underwriters corporation;

xanthohumol (Xanthohumol) was purchased from Selleck corporation.

EXAMPLE 1 determination of the cytotoxicity of xanthohumol on Vero-E6 cells

Vero-E6 cells were counted, diluted to appropriate density with DMEM nutrient solution containing 8% Fetal Bovine Serum (FBS) and then diluted to 1.5 × 10⁴The concentration of each well was measured in a 96-well plate at 37 ℃ with 5% CO₂After the cells adhere to the monolayer in the incubator (about 18-20h), the xanthohumol is diluted with DMEM nutrient solution to: 1 μ M, 2 μ M, 5 μ M, 10 μ M, 3 replicates per concentration setting. DMSO pairs were plated on 96-well cell culture platesTreating Vero-E6 cells with diluted xanthohumol sample at a concentration of 100 μ l/well for 72h, removing supernatant, adding 90 μ l fresh culture solution, adding 10 μ l MTT solution, and culturing for 4 h; and (4) absorbing and removing the supernatant, adding 110 mu l of formazan dissolving solution into each hole, and placing the mixture on a shaking table to shake for 10min at a low speed to fully dissolve the crystals. The absorbance of each well was measured at 490nm in an ELISA detector, and the results are shown in FIG. 1.

As can be seen from FIG. 1, there was no significant difference in the amount of mitochondrial dehydrogenase in cells treated with different concentrations of xanthohumol compared to cells not treated with xanthohumol, indicating that this concentration of xanthohumol was not significantly cytotoxic on Vero-E6 cells.

EXAMPLE 2Western Blot assay xanthohumol inhibits porcine epidemic diarrhea Virus infection on Vero-E6 cells Activity of (2)

Vero-E6 cells were counted, diluted to appropriate density with DMEM nutrient solution containing 8% Fetal Bovine Serum (FBS) and then diluted to 7 × 10⁵The concentration of each well was adjusted to 6-well plates and placed at 37 ℃ in 5% CO₂After the cells adhered to form a monolayer and grown to a density of 70-80% (about 18-20H) in the incubator, the Vero-E6 cells were pretreated with DMSO control and xanthohumol at different concentrations for 2H at 37 ℃ by diluting xanthohumol with DMEM nutrient solution to 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, infecting with porcine epidemic diarrhea virus H L JBY strain (MOI 0.01), and placing at 37 ℃ with 5% CO₂Washing with PBS for 1 hr in incubator for three times, adding 1ml DMEM containing 2% Fetal Bovine Serum (FBS) and xanthohumol, standing at 37 deg.C and 5% CO₂After 24 hours of culture in an incubator, a cell sample is collected by using 2 × SDS samplebuffer, the cell sample is subjected to metal bath boiling for 10 minutes, Western Blot detection is carried out, and the result is shown in figure 2, which shows that the xanthohumol can obviously inhibit the infection of porcine epidemic diarrhea virus and has obvious dose dependence.

Example 3 fluorescent quantitation of xanthohumol Activity against porcine epidemic diarrhea Virus infection on Vero-E6 cells Property of (2)

Vero-E6 cells were counted, diluted to appropriate density with DMEM nutrient solution containing 8% Fetal Bovine Serum (FBS) and then diluted to 7 × 10⁵The concentration of each well was adjusted to 6-well plates and placed at 37 ℃ in 5% CO₂Wait in incubatorAfter the cells adhered to form a monolayer and grown to a density of 70-80% (about 18-20H), xanthohumol was diluted to 10 μm with DMEM nutrient solution, the Vero-E6 cells were pretreated with DMSO control and xanthohumol at 37 ℃ for 2H, infected with porcine epidemic diarrhea virus H L JBY strain (MOI ═ 0.01), and placed at 37 ℃ with 5% CO₂Washing with PBS for 1 hr in incubator for three times, adding 1ml DMEM containing 2% Fetal Bovine Serum (FBS) and xanthohumol, standing at 37 deg.C and 5% CO₂After 24h incubation in the incubator, cell samples were collected using Trizon and RNA was extracted. The extracted RNA was dissolved in 20. mu.l of ultrapure water, inverted to cDNA, and the change in the mRNA level of the PEDV-M protein was detected by real-time fluorescent quantitative PCR using the Actin gene as an internal reference. Wherein the primer information is as follows:

Actin-F 5’CTGAAGTACCCCATCGAGCACGGCA’3’；

Actin-R 5’GGATAGCACAGCCTGGATAGCAACG’3’；

PEDV-M-F 5’CCCGTTGATGAGGTGATTG 3’；

PEDV-M-R 5’CGAAAAAGACCCAGTTGACC 3’。

20 ul PCR reaction containing 10 ul AceQ

Green Master Mix, 0.4. mu.l upstream and downstream primers (10. mu.M), 2. mu.l template DNA, 0.4. mu.l ROX Reference Dye1 and 6.8. mu.l sterile distilled water. The amplification parameters were: 40 cycles of 95 ℃ 10s and 60 ℃ 30s, and after the reaction is finished, the dissolution curve is measured, and the reaction conditions are as follows: 95 ℃ for 15s, 60 ℃ for 60s and 95 ℃ for 15 s. This experiment was carried out by 2^-ΔΔCtThe method carries out relative quantification of the PEDV-M gene, the result is shown in figure 3, and the activity of xanthohumol for obviously inhibiting the porcine epidemic diarrhea virus infection on Vero-E6 cells is shown.

EXAMPLE 4 plaque formation assay determination of xanthohumol inhibition of porcine epidemic diarrhea Virus infection on Vero-E6 cells Activity of (2)

Vero-E6 cells were counted, diluted to appropriate density with DMEM nutrient solution containing 8% Fetal Bovine Serum (FBS) and then diluted to 7 × 10⁵The concentration of each well was adjusted to 6-well plates and placed at 37 ℃ in 5% CO₂When the cells adhere to the wall to form a monolayer and grow to 70-80% in the culture boxAfter the density (about 18-20H), the xanthohumol was diluted with DMEM nutrient solution to 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, Vero-E6 cells were pretreated with DMSO control and different concentrations of xanthohumol at 37 ℃ for 2H, infected with porcine epidemic diarrhea virus H L JBY strain (MOI 0.01), placed at 37 ℃ and 5% CO₂Washing with PBS for 1 hr in incubator for three times, adding 1ml DMEM containing 2% Fetal Bovine Serum (FBS) and xanthohumol, standing at 37 deg.C and 5% CO₂Culturing in incubator for 24 hr, collecting supernatant, diluting, inoculating to 6-well plate with monolayer cells, placing at 37 deg.C and 5% CO₂Washing with PBS for 1 hr for three times in incubator, adding 2ml of 2 × DMEM containing 4% fetal calf serum and 2% low melting point agarose 1: 1 mixture, 37 deg.C, and 5% CO₂Culturing in incubator for 2-3 days, observing plaque formation, adding 1% crystal violet when obvious visible plaque appears, and staining, and the result is shown in figure 4, which shows that xanthohumol can obviously inhibit the infection of porcine epidemic diarrhea virus and has obvious dose dependence.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention should not be limited by the disclosure of the preferred embodiments. Therefore, it is intended that all equivalents and modifications which do not depart from the spirit of the invention disclosed herein are deemed to be within the scope of the invention.

Claims (3)

1. The application of the xanthohumol as the only active ingredient in the preparation of the medicine for resisting the porcine epidemic diarrhea virus is that the application dose of the xanthohumol is 1-10 mu M/L, and the porcine epidemic diarrhea virus is a porcine epidemic diarrhea virus H L JBY strain.

2. The use of claim 1, wherein the medicament for resisting porcine epidemic diarrhea virus comprises xanthohumol and pharmaceutically acceptable auxiliary materials or auxiliary components, wherein the mass percentage content of the xanthohumol is not less than 99.75%, and the application dose is 1-10 μ M/L.

3. The use of claim 2, wherein the anti-porcine epidemic diarrhea virus medicament is in a dosage form selected from the group consisting of a tablet, a powder, a granule, a capsule, an oral liquid, an injection, and a sustained release formulation.

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