CN111110778B

CN111110778B - Application of aloe extract in preparation of medicine for preventing and treating porcine epidemic diarrhea

Info

Publication number: CN111110778B
Application number: CN202010079060.2A
Authority: CN
Inventors: 曹永长; 胡文锋; 刘元; 徐志超; 靳改改; 吴雨; 胡学生
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2020-02-03
Filing date: 2020-02-03
Publication date: 2021-10-01
Anticipated expiration: 2040-02-03
Also published as: CN111110778A

Abstract

The invention relates to application of aloe extract in preparing a medicament for preventing and treating porcine epidemic diarrhea. The aloe extract preparation and PEDV are mixed and inoculated into Vero and IPEC-J2 cells, and after cytopathic effect is detected by using an indirect immunofluorescence assay (IFA), a protein immunoblotting assay (WB) and a virus titer determination method, the result shows that the aloe extract can effectively inhibit the proliferation of PEDV in Vero cells and IPEC-J2 cells, has a dose-dependent effect, and can completely inhibit the proliferation of PEDV under a high dose condition (16 mg/ml).

Description

Application of aloe extract in preparation of medicine for preventing and treating porcine epidemic diarrhea

Technical Field

The invention relates to the field of biotechnology application, in particular to application of a natural plant extract in preparation of a medicament for preventing and treating porcine epidemic diarrhea.

Background

Porcine Epidemic Diarrhea (PED) is an acute, highly infectious disease of pigs whose pathogenic Porcine Epidemic Diarrhea Virus (PEDV) is a single-stranded positive-strand RNA Virus with an envelope belonging to the genus Alphacoronavirus (KOCHERRANS R, BRIDGEN A, ACKERMANN M, et al.2001.completion of the Porcine epidemic diarrheea coronavirus (PEDV) genome sequence [ J ] Virus Genes,23(2): 137-. PEDV can cause diarrhea, dehydration and vomiting of newborn piglets, and has high fatality rate.

In recent years, PEDs have been widely spread throughout the world, and countries such as china, the united states, korea, and canada have been in outbreak of epidemic situations, which seriously jeopardize the development of the global pig industry. 29 provinces and cities of China occur and prevail PED in autonomous regions, so that the death rate of newborn piglets can reach 100 percent, and huge economic loss is caused to the pig raising industry in China.

At present, the prevention and control of the disease are mainly based on vaccine immunization prevention and control. Vaccine immunization is based on direct immunization of piglets, and vaccine immunization protects intestinal epithelial cells by inducing mucosal immunity (WANG D, FANG L, XIAO S.2016. hormone epidemic diarrhea in China [ J ]. Virus Res,226: 7-13.). For newborn piglets and lactating piglets, there are two problems with vaccine immunization: first, sows whose serum is positive for PEDV antibodies, maternal antibodies in milk, will affect the protective effect induced by oral live vaccines; secondly, the PEDV vaccine plays a role through mucosal immunity, but high-incidence groups of PED are newborn piglets and suckling-period piglets with incompletely developed mucosal immune systems, so that the vaccine immune effect of the PEDV is not ideal.

In addition to vaccine immunization, chemically synthesized drugs, hormones, antibiotics, etc. are currently used to alleviate diarrhea symptoms. In addition, the plant components have the advantages of no drug residue and no pollution, and become a popular antiviral research direction at present. For example, Liu Yan fen, et al, Heilongjiang animal husbandry and veterinary (2016,18:155-⁺298) A paper entitled research on treatment effect of a traditional Chinese medicine compound on piglets infected with porcine epidemic diarrhea viruses is published, and a paper entitled research on treatment effect of compound traditional Chinese medicine oral liquid on piglet diarrhea is published in the journal of Chinese veterinary medicine (2015,02:55-56.) by New York et al, and a paper entitled research on treatment effect of a self-prepared traditional Chinese medicine compound on porcine epidemic diarrhea is published in the veterinary medicine of Heilongjiang (2014(22):99-100.) by Yibao Ying. The research shows that the compound traditional Chinese medicine can effectively treat diarrhea caused by PEDV, and has obvious antiviral effect proved by in vitro experiments.

Although the traditional Chinese medicine compound has the effect of treating the porcine epidemic diarrhea, a plurality of Chinese herbal medicines are required to be compatible for use, the components are complex, and the preparation steps are complicated.

Disclosure of Invention

In view of the above, the invention provides a plant extract with a single component and an effect of inhibiting porcine epidemic diarrhea virus, which can be used for treating porcine epidemic diarrhea and related medicines, aiming at the problems of complex components, complicated preparation steps and the like of a traditional Chinese medicine compound.

Application of Aloe extract in preparing medicine for preventing and treating porcine epidemic diarrhea is provided.

The aloe extract is prepared by the following method: decocting the aerial parts of Aloe with water to obtain paste, or extracting with water and ethanol, lyophilizing, and pulverizing to obtain powder.

The aloe extract comprises the following main effective components: aloe polysaccharides (dextran, glucomannan, etc.), anthraquinones (such as aloin, aloe-emodin, chrysophanol, and aloe saponin, etc.). The literature reports that aloe extracts have the following functions:

(1) sterilizing and anti-inflammatory

Most of anthrone compounds in the aloe have the effects of sterilizing, inhibiting bacteria, diminishing inflammation, detoxifying, promoting wound healing and the like, can effectively eliminate acne and acne, is clinically used for treating various inflammations, and has obvious curative effect.

(2) Strengthening stomach and relieving diarrhea

Aloe has effects of invigorating stomach and relieving diarrhea, and is mainly due to the fact that Aloe contains effective components such as aloe-emodin glycoside, aloenin, and aloe tincture. Has effects in invigorating stomach, relieving diarrhea, stimulating appetite, and relieving diarrhea in large intestine. Has a main action part in large intestine, and can be used for treating constipation.

(3) Anticancer medicine

Researches show that aloe gel, a sticky substance contained in aloe, has strong anticancer effect. The anti-tumor research of aloe in China discovers that the aloe alcohol extract and aloesin can obviously improve the immune function of organisms, and aloe is clinically applied to treat liver cancer, gastric cancer and the like.

(4) Beauty treatment

The aloin contained in Aloe has skin beautifying effect, and has effects in keeping moisture, relieving inflammation, inhibiting bacteria, relieving itching, resisting allergy, softening skin, preventing acne, inhibiting sweat, deodorizing, etc., and can strongly absorb ultraviolet ray to prevent skin burn.

(5) Enhancing immunity and improving sub-health status

Aloe polysaccharide can regulate immunity, and has effect in improving autoimmunity and resisting bacteria. The aloin and polysaccharide can strengthen heart function, promote blood circulation, soften hardened artery, reduce cholesterol value, dilate capillary vessel, normalize blood pressure, and prevent and treat arteriosclerosis and hypertension.

Owing to the functions, the aloe extract has wide application. However, the efficacy of aloe extracts in preventing and treating porcine epidemic diarrhea and medicaments thereof are not reported in comprehensive domestic and foreign research.

The application of the aloe extract in preparing the medicine for preventing and treating the porcine epidemic diarrhea can inhibit the porcine epidemic diarrhea virus by using the aloe extract, and has the advantages of single and simple component, mature preparation method and wide raw materials. The aloe extract is a pure plant extract, has no toxic or side effect, and can improve the innate immunity and virus resistance of organisms and directly kill viruses, thereby preventing and treating porcine epidemic diarrhea.

Drawings

FIG. 1 shows the effect of aloe vera extract (Ae) on Vero cell activity.

FIG. 2 shows the results of IFA inhibition of PEDV-N protein expression in Vero cells by different concentrations of aloe vera extract (Ae).

FIG. 3 shows the WB results of different concentrations of Aloe vera extract (Ae) inhibiting the expression of PEDV-N protein in Vero cells at different time points.

FIG. 4 shows the results of virus titer assay for PEDV titer at different concentrations of aloe vera extract (Ae) for different periods of time.

FIG. 5 shows the effect of aloe vera extract on IPEC-J2 cell activity.

FIG. 6 is the IFA results of various concentrations of aloe vera extract (Ae) inhibiting the expression of PEDV-N protein in IPEC-J2 cells.

FIG. 7 is the WB results of different concentrations of aloe vera extract (Ae) inhibiting the expression of PEDV-N protein in IPEC-J2 cells at different time points.

FIG. 8 shows the results of virus titer assay for PEDV titer at different concentrations of aloe vera extract (Ae) for different periods of time.

Detailed Description

The present invention will be described in further detail with reference to examples.

The aloe extracts used in the examples of the present invention were prepared by the Cobble and Coats heat treatment process. The aloe extract of the present invention may also be prepared by any of the methods disclosed in the prior art for preparing aloe extracts or commercially available aloe extracts.

The assay methods used in the present invention are those conventionally used in the art, and the assay reagents, cells, etc. used therein are commercially available or prepared according to the conventional methods in the art.

Example 1 Aloe extract assay for Vero cytotoxicity and inhibition of PEDV proliferation in Vero cells

1.1 Aloe Extract (AE) assay for Vero cytotoxicity detection

Inoculation of 6X 10 in 96-well plates⁵cells/mL cell suspension (100. mu.L/well) leaving a column without seeding cells. At 37 ℃ with 5% CO₂Culturing under the condition until the cells are completely confluent. The medium was aspirated, washed 3 times with PBS, and aloe vera extract diluted to different concentrations with DMEM and added to 96-well plates (100. mu.L/well) with 8 replicates per dilution. At 37 ℃ with 5% CO₂Culturing under the conditions of 24 hr and 48 hr, removing the medicinal liquid, washing with PBS for 3 times, adding 10% CCK-8 solution into 96-well plate (100 μ L/well) with DMEM, and culturing at 37 deg.C with 5% CO₂Incubating for 1h under the condition, measuring an absorbance value (lambda is 450nm) by using a microplate reader, and calculating the cell viability after treating for 24h and 48h by using liquid medicine with different concentrations.

The results are shown in fig. 1, where the maximum safe concentration of aloe extract to Vero cells was 16mg/mL (n-8).

1.2IFA measurement of the intensity of the PEDV-N protein fluorescence after Vero cells have been treated with different concentrations of Aloe Extract (AE)

1. In 12-well cell culture plates at 6X 10⁵cells/mL cell concentration 1mL cell suspension, 6 wells at 37 ℃, 5% CO₂Culturing under the condition until the cells are completely confluent.

2. Washing culture medium in discarded 12-well plate with PBS for 3 times, adding DMEM culture medium with aloe extract concentration of 0mg/mL (negative control group), 0mg/mL (positive control group), 2mg/mL, 4mg/mL, 8mg/mL and 16mg/mL into 6 wells with cells, respectively, treating for 1h, discarding the treated solution, adding aloe extract treated solution with PEDV (MOI of 0.2) with corresponding concentration, treating the first aloe extract with 0mg/mL as negative control without virus inoculation, treating for 1h, discarding the treated solution, washing with PBS for 3 times, adding aloe extract treated solution without PEDV with corresponding concentration, washing with PBS for 3 times, and treating at 37 deg.C with 5% CO₂And culturing for 24 hours under the condition.

3. Discard the treatment solution, wash with PBST 3 times for 5min each time, add 4% paraformaldehyde precooled at 4 deg.C, 500. mu.L per well, incubate at room temperature for 15 min.

4. Washing with PBST for 3 times, 5min each time, adding 0.4% Triton X-100 solution, and incubating at room temperature for 10-20 min.

5. Wash 3 times with PBST for 5min each, add 3% BSA, 500 μ L per well, and incubate at room temperature for 1 h.

6. Wash 3 times with PBST for 5min each, dilute 1D11 primary antibody with PBST solution instrument containing 1% BSA at a ratio of 1:1000, 300 μ L per well, incubate for 1h at 37 ℃.

7. The primary anti-dilution solution 1D11 was recovered, washed 3 times with PBST for 5min each time, and FITC fluorescent secondary antibody was diluted with PBST at a ratio of 1:500, 300. mu.L per well, and incubated at 37 ℃ for 1h in the absence of light.

8. Washing with PBST for 3 times (5 min each) in the dark, diluting DAPI fluorescent dye with PBST at a ratio of 1:1000, incubating at 37 deg.C in the dark for 7min and 300 μ L per well.

9. Washing with PBST for 5-7 times in dark condition, supplementing PBST 500 μ L to each well, observing fluorescence intensity with fluorescence microscope and taking pictures.

As shown in FIG. 2, the proliferation of PEDV virus after aloe extract treatment was measured by indirect immunofluorescence assay (IFA), and the specific fluorescence of PEDV-N protein gradually decreased after 24h drug treatment as the drug concentration increased.

1.3WB detection of the Presence or absence of PEDV-N protein bands in Vero cells treated with different concentrations of Aloe extract

1. In 3 12-well cell culture plates at 6X 10⁵cells/mL cell concentration 1mL cell suspension, each 12-well plate 6 wells, at 37 degrees C, 5% CO₂Culturing under the condition until the cells are completely confluent.

2. Washing culture medium in discarded 12-well plate with PBS for 3 times, adding DMEM culture medium with aloe extract concentration of 0mg/mL (negative control group), 0mg/mL (positive control group), 2mg/mL, 4mg/mL, 8mg/mL and 16mg/mL into 6 wells with cells, respectively, treating for 1h, discarding the treated solution, adding aloe extract treated solution containing PEDV (MOI 0.2), treating the first aloe extract with 0mg/mL as negative control, not inoculating, treating for 1h, discarding the treated solution, washing with PBS for 3 times, adding aloe extract treated solution containing no PEDV, and treating at 37 deg.C and 5% CO for 3 times₂Culturing for 12h, 24h and 48h respectively.

3. Discarding the treatment solution, washing with PBS for 3 times, adding 100. mu.L RIPA lysate (Weak) into each well, lysing for 10min on ice, blowing and mixing cell precipitates uniformly, transferring to a 1.5mL sterile EP tube, adding 25. mu.L 5 × Sample Buffer into each tube, vortexing for 5s, water-bathing for 10min at 100 ℃, and centrifuging for 1min at 12000 r/min.

4. Electrophoresis was performed with 12-well, 10% pre-gel, in the order: PBS, Marker, 0mg/mL (negative control group), 0mg/mL (positive control group), 2mg/mL, 4mg/mL, 8mg/mL, 16mg/mL, PBS, and electrophoresed for 40-60min with 150V voltage.

5. According to the sizes of a target protein PEDV-N (55kDa) and an internal reference protein GAPDH (35kDa), the gel is cut at 25kDa and 75kDa respectively, the gel is cut at the left and right from a PBS lane, and the cut gel is transferred into a precooled transfection solution.

6. And soaking the cut PVDF membrane in precooled methanol for 1min, transferring into precooled membrane transferring liquid, and soaking the cut filter paper in the precooled membrane transferring liquid.

7. The assembly is changeed the membrane splint, and the assembly order is: and removing air bubbles by using a clean test tube or a glass rod after assembling the black surface (cathode), the sponge, the filter paper, the protein glue, the PVDF membrane, the filter paper, the sponge and the white surface (anode), and putting the assembled clamping plate into a transfer printing groove to be completely immersed in the transfer printing liquid. The transfer tank was placed in an ice bath at 100V/300mA for 1 h.

8. After the transfer, the PVDF membrane was removed, washed with TBST for 5min, placed in 3% BSA blocking solution, and blocked overnight at 4 ℃.

9. Pouring off the blocking solution, washing with PBST for 3 times, 5min each time, placing on a shaking bed for 60r/min, taking out the membrane, wrapping with a clean preservative film, cutting the membrane from a 45kDa Marker, placing the membrane with the 45-75kDa protein in PEDV-N polyclonal antibody solution diluted by TBST containing 1% BSA (dilution ratio 1:1000), placing the membrane with the 25-45kDa protein in GAPDH monoclonal antibody solution diluted by TBST containing 1% BSA (dilution ratio 1:1000), placing on the shaking bed, 20r/min, and incubating at room temperature for 1 h.

10. Respectively recovering PEDV-N polyclonal antibody diluent and GAPDH monoclonal antibody diluent, washing the membrane with PBST for 3 times, each time for 5min, placing the membrane on a shaking table for 60r/min, placing the two membranes in HRP-labeled goat anti-mouse IgG secondary antibody solution diluted by TBST (the dilution ratio is 1:8000), placing the membrane on the shaking table for 20r/min, and incubating at room temperature for 1 h.

11. And recovering the secondary antibody diluent, washing the membrane for 3 times by using TBST (tunnel boring machine) for 5min each time, placing the membrane on a shaking table for 60r/min, soaking the membrane in the TBST, preparing an ECL (electron cyclotron resonance) color developing solution under the condition of keeping out of the sun, and developing and photographing by using a gel scanning system.

As shown in FIG. 3, the proliferation of PEDV after the aloe extract treatment was examined by Western Blotting (WB), and the color of the band corresponding to the size of PEDV-N protein gradually decreased with the increase of the drug concentration after the drugs were treated for 12h, 24h, and 48h, respectively.

1.4 detection of PEDV titre in Vero cells treated with different concentrations of Aloe extract Using Virus titre assay

1. In 3 12-well cell culture plates at 6X 10⁵cell/mL cell concentration 1mL cells per well, 4 wells per 12-well plate, 5% CO at 37 ℃%₂Culturing under the condition until the cells are completely confluent.

2. Culture in discarded 12-well platesWashing with PBS for 3 times, adding DMEM medium with aloe extract concentration of 0mg/mL (negative control group), 0mg/mL (positive control group), 8mg/mL and 16mg/mL into 4 wells with cells, treating for 1 hr, discarding the treated solution, adding aloe extract treated solution containing PEDV (MOI 0.2), treating the first aloe extract well with 0mg/mL as negative control without virus inoculation for 1 hr, discarding the treated solution, washing with PBS for 3 times, adding aloe extract treated solution containing no PEDV at the corresponding concentration, washing with 5% CO at 37 deg.C₂Culturing for 12h, 24h and 48h respectively.

3. Collecting samples according to time points, sealing 12-hole plates at different time points with sealing films, freezing and thawing at-80 deg.C for 3 times, subpackaging the frozen and thawed culture in sterile 1.5mL EP tube, and preserving at-80 deg.C.

4. According to 6X 10⁵cells/mL cell concentration in 96-well plates, 100. mu.L per well, at 37 ℃ with 5% CO₂Culturing under the condition until the cells are completely confluent, washing the 96-well plate with grown cells with PBS 3 times, diluting the collected sample by 10 times for 5 gradients, each gradient being 4 times, adding the diluted sample into corresponding cell wells at a concentration of 100 μ L per well, and culturing at 37 deg.C and 5% CO₂Culturing under the condition for 5-7 days.

5. The number of lesion wells for each dilution gradient was recorded under microscope observation and the number of viable virus particles of PEDV treated with different concentrations of aloe extract at different time points was calculated.

As shown in fig. 4, proliferation of PEDV virus after aloe extract treatment was measured by using virus titer assay, and the virus titer assay after the same treatment showed that PEDV titer was reduced to 0pfu/ml (n-4) after the drug reached a certain concentration.

Example 2 Aloe extract cytotoxicity assay on IPEC-J2 and assay for inhibiting the proliferation of PEDV in IPEC-J2 cells

2.1 Aloe Extract (AE) cytotoxicity assay on IPEC-J2

Inoculation of 6X 10 in 96-well plates⁵cells/mL cell suspension (100. mu.L/well) leaving a column without seeding cells. At 37 ℃ with 5% CO₂Culturing under the condition until the cells are completely confluent. The culture medium is aspirated, PBS is washed for 3 times, and the drug to be tested is addedThe material (aloe extract (AE)) was diluted to different concentrations with DMEM and added to 96-well plates (100 μ L/well) with 8 replicates per dilution. Culturing at 37 deg.C under 5% CO2 for 24 hr and 48 hr, removing the liquid medicine, washing with PBS for 3 times, adding 10% CCK-8 solution into 96-well plate (100 μ L/well) with DMEM, and culturing at 37 deg.C under 5% CO2₂Incubating for 1h under the condition, measuring an absorbance value (lambda is 450nm) by using a microplate reader, and calculating the cell viability after treating for 24h and 48h by using liquid medicine with different concentrations.

The results are shown in fig. 5, where the maximum safe concentration of aloe extract to IPEC-J2 cells was 16mg/mL (n-8).

2.2IFA determination of the intensity of the PEDV-N protein fluorescence after IPEC-J2 cells treated with different concentrations of Aloe extract

1. In 12-well cell culture plates at 6X 10⁵cells/mL cell concentration 1mL cell suspension per well, 5 wells at 37 deg.C with 5% CO₂Culturing under the condition until the cells are completely confluent.

2. Washing culture medium in discarded 12-well plate with PBS for 3 times, adding DMEM culture medium with aloe extract concentration of 0mg/mL (negative control group), 0mg/mL (positive control group), 4mg/mL, 8mg/mL and 16mg/mL into 5 wells with cells, respectively, treating for 1h, discarding the treated solution, adding aloe extract treated solution containing PEDV (MOI 0.4), treating the first aloe extract well with 0mg/mL as negative control, not inoculating virus, treating for 1h, discarding the treated solution, washing with PBS for 3 times, adding aloe extract treated solution containing no PEDV, treating at 37 deg.C and 5% CO for 3 times₂And culturing for 24 hours under the condition.

7. The primary anti-dilution solution of 1D11 was recovered, washed 3 times with PBST for 5min each time, and Cy3 fluorescent secondary antibody was diluted with PBST at a ratio of 1:500, 300. mu.L per well, and incubated for 1h at 37 ℃ in the absence of light.

As shown in FIG. 6, the proliferation of PEDV virus after aloe extract treatment was measured by indirect immunofluorescence assay (IFA), and the specific fluorescence of PEDV-N protein gradually decreased after 24h drug treatment as the drug concentration increased.

2.3WB detection of the Presence or absence of PEDV-N protein bands on IPEC-J2 cells treated with different concentrations of Aloe extract

1. In 3 12-well cell culture plates at 6X 10⁵cell/mL cell concentration 1mL cell suspension per well, 5 wells per 12-well plate, 5% CO at 37 ℃%₂Culturing under the condition until the cells are completely confluent.

2. Washing culture medium in discarded 12-well plate with PBS for 3 times, adding DMEM culture medium with aloe extract concentration of 0mg/mL (negative control group), 0mg/mL (positive control group), 4mg/mL, 8mg/mL and 16mg/mL into 5 wells with cells, respectively, treating for 1h, discarding the treated solution, adding aloe extract treated solution containing PEDV (MOI 0.4), treating the first aloe extract well with 0mg/mL as negative control, not inoculating virus, treating for 1h, discarding the treated solution, washing with PBS for 3 times, adding aloe extract treated solution containing no PEDV, treating at 37 deg.C and 5% CO for 3 times₂Culturing for 12h, 24h and 48h respectively.

4. Electrophoresis was performed with 12-well, 10% pre-gel, in the order: PBS, Marker, 0mg/mL (negative control group), 0mg/mL (positive control group), 4mg/mL, 8mg/mL, 16mg/mL, PBS, and electrophoresed for 40-60min with 150V voltage.

9. Pouring the blocking solution, washing with TBST for 3 times, 5min each time, placing on a shaking bed for 60r/min, taking out the membrane, wrapping with a clean preservative film, cutting the membrane from a 45kDa Marker, placing the membrane with the 45-75kDa protein in PEDV-N polyclonal antibody solution diluted by TBST containing 1% BSA (dilution ratio 1:1000), placing the membrane with the 25-45kDa protein in GAPDH monoclonal antibody solution diluted by TBST containing 1% BSA (dilution ratio 1:1000), placing on the shaking bed, 20r/min, and incubating at room temperature for 1 h.

10. Respectively recovering PEDV-N polyclonal antibody diluent and GAPDH monoclonal antibody diluent, washing the membrane with TBST for 3 times, each time for 5min, placing the membrane on a shaking table for 60r/min, placing the two membranes in HRP-labeled goat anti-mouse IgG secondary antibody solution diluted with TBST (the dilution ratio is 1:8000), placing the membrane on the shaking table, performing incubation for 1h at room temperature at 20 r/min.

As shown in FIG. 7, the proliferation of PEDV after the aloe extract treatment was examined by Western Blotting (WB), and the color of the band corresponding to the size of PEDV-N protein gradually decreased with the increase of the drug concentration after the drugs were treated for 12h, 24h, and 48h, respectively.

3.4 detection of the titer of PEDV in IPEC-J2 cells treated with different concentrations of Aloe extract by Virus titer assay

1. In 3 12-well cell culture plates at 6X 10⁵cell/mL cell concentration 1mL cells per well, 5 wells per 12-well plate, 5% CO at 37 ℃%₂Culturing under the condition until the cells are completely confluent.

2. Washing culture medium in discarded 12-well plate with PBS for 3 times, adding DMEM culture medium with aloe extract concentration of 0mg/mL (negative control group), 0mg/mL (positive control group), 4mg/mL, 8mg/mL and 16mg/mL into 6 wells with cells, respectively, treating for 1h, discarding the treated solution, adding aloe extract treated solution containing PEDV (MOI 0.4), treating the first aloe extract well with 0mg/mL as negative control, not inoculating virus, treating for 1h, discarding the treated solution, washing with PBS for 3 times, adding aloe extract treated solution containing no PEDV, treating at 37 deg.C and 5% CO for 3 times₂Culturing for 12h, 24h and 48h respectively.

4. According to 6X 10⁵cells/mL cell concentration in 96-well plate, 100 μ L per well, at 37 deg.C and 5% CO2 condition culture until cells completely confluent, washing the well-grown 96-well plate with PBS 3 times, diluting the collected sample by 10 times with 5 gradients, each gradient is repeated 4 times, adding the diluted sample into corresponding cell well, 100 μ L per well, at 37 deg.C and 5% CO 4₂Culturing under the condition for 5-7 days.

5. The number of lesion wells for each dilution gradient was recorded under microscope observation and the titer of PEDV was calculated at different time points and after treatment with different concentrations of aloe vera extract.

As shown in fig. 8, proliferation of PEDV virus after aloe extract treatment was measured by using virus titer assay, and the virus titer assay after the same treatment showed that PEDV titer was reduced to 0pfu/ml (n ═ 4) after the drug reached a certain concentration.

The new application of the aloe extract can be used for inhibiting replication of the enveloped virus PEDV, and the aloe extract can be used for preventing and treating porcine epidemic diarrhea caused by PEDV infection, and further can be applied to preparation of medicaments for preventing and treating porcine epidemic diarrhea caused by PEDV infection.

The aloe extract preparation and PEDV are mixed and inoculated into Vero and IPEC-J2 cells, and after cytopathic effect is detected by using an indirect immunofluorescence assay (IFA), a protein immunoblotting assay (WB) and a virus titer determination method, the result shows that the aloe extract can effectively inhibit the proliferation of PEDV in Vero cells and IPEC-J2 cells, has a dose-dependent effect, and can completely inhibit the proliferation of PEDV under a high dose condition (16 mg/ml).

The above examples are merely representative of a few embodiments of the present invention, and although the description is specific and detailed, the present invention should not be construed as limited the scope of the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. The application of aloe extract in preparing the medicine for preventing and treating porcine epidemic diarrhea is characterized in that the aloe extract is prepared by the following method: decocting the aerial parts of Aloe with water to obtain paste, or extracting with water and ethanol, lyophilizing, and pulverizing to obtain powder.