CN115068452B - Application of aloe-emodin in preparing medicine for preventing and treating African swine fever - Google Patents
Application of aloe-emodin in preparing medicine for preventing and treating African swine fever Download PDFInfo
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- CN115068452B CN115068452B CN202210256584.3A CN202210256584A CN115068452B CN 115068452 B CN115068452 B CN 115068452B CN 202210256584 A CN202210256584 A CN 202210256584A CN 115068452 B CN115068452 B CN 115068452B
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- emodin
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- YDQWDHRMZQUTBA-UHFFFAOYSA-N Aloe emodin Chemical compound C1=CC=C2C(=O)C3=CC(CO)=CC(O)=C3C(=O)C2=C1O YDQWDHRMZQUTBA-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 208000007407 African swine fever Diseases 0.000 title claims abstract description 17
- 239000003814 drug Substances 0.000 title claims abstract description 14
- 241000701386 African swine fever virus Species 0.000 claims abstract description 31
- 150000003839 salts Chemical class 0.000 claims description 5
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 4
- 239000012752 auxiliary agent Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 15
- 229940079593 drug Drugs 0.000 abstract description 4
- 241000710777 Classical swine fever virus Species 0.000 abstract description 2
- 239000004480 active ingredient Substances 0.000 abstract description 2
- 230000001681 protective effect Effects 0.000 abstract description 2
- 231100000673 dose–response relationship Toxicity 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 41
- 241000700605 Viruses Species 0.000 description 11
- 239000000243 solution Substances 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 8
- 101710120319 Photosystem I reaction center subunit IV Proteins 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229930014626 natural product Natural products 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 230000001464 adherent effect Effects 0.000 description 5
- 238000010609 cell counting kit-8 assay Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000003556 assay Methods 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000263 cytotoxicity test Toxicity 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 101710132601 Capsid protein Proteins 0.000 description 3
- 101710189818 Non-structural protein 2a Proteins 0.000 description 3
- 101710151911 Phosphoprotein p30 Proteins 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 241001116389 Aloe Species 0.000 description 2
- 210000003771 C cell Anatomy 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 235000011399 aloe vera Nutrition 0.000 description 2
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
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- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- 238000010257 thawing Methods 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
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- 241000283707 Capra Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 240000004980 Rheum officinale Species 0.000 description 1
- 235000008081 Rheum officinale Nutrition 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 210000001132 alveolar macrophage Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- -1 anthraquinone compound Chemical class 0.000 description 1
- 230000003527 anti-angiogenesis Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses an application of aloe-emodin in preparing a medicine for preventing and treating African swine fever, and belongs to the field of veterinary medicines. The aloe-emodin has remarkable protective effect on cells infected with African swine fever virus, has remarkable anti-African swine fever virus activity and is in a dose-dependent relationship. The aloe-emodin can be used as an active ingredient for preparing a novel medicine for preventing and treating African swine fever, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of veterinary medicines, and particularly relates to application of aloe-emodin in preparation of medicines for preventing and treating African swine fever.
Background
African swine fever (African swine fever, ASF) is an epidemic infectious disease caused by African swine fever virus (African swine fever virus, ASFV). First in kennia in the eastern africa in the 20 th century, and later spread to europe, south america, asia, etc.
Aloe-emodin is an anthraquinone compound extracted from aloe leaves, is an active ingredient of many Chinese herbal medicines, such as rheum officinale, aloe and semen cassiae, and has been found to have many pharmacological activities, such as anti-tumor, anti-oxidation, anti-cancer and anti-angiogenesis. In addition, aloe-emodin has also been shown to have a broad antiviral effect. The chemical name of aloe-emodin is 1.8-dihydroxy-3-hydroxymethyl anthraquinone, and the molecular formula is as follows: c (C) 15 H 10 O 5 Relative molecular mass: 270.2369 the chemical structural formula of aloe-emodin is as follows.
At present, research reports on the anti-African swine fever virus effect of aloe-emodin are not found, and related patents are not found.
Disclosure of Invention
The invention surprisingly discovers that the natural product aloe-emodin can obviously inhibit the expression of ASFV structural protein p30, thereby preventing viruses from infecting host cells and playing a role in inhibiting ASFV infection.
The invention aims to provide an application of aloe-emodin as a natural product in preparing an ASFV-resisting medicament and an African swine fever-preventing medicament.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
firstly, detecting toxicity of aloe-emodin to Pig Alveolar Macrophages (PAM) by using a CCK-8 (Cell Counting Kit-8) method, and determining the safe working concentration of the aloe-emodin. Subsequently, the protective effect of aloe-emodin on PAM cells infected with ASFV was evaluated using the established ASFV cell infection model. The evaluation mode is mainly fluorescence quantitative PCR (qPCR) test and half red blood cell adsorption (HAD) 50 ) Assays, indirect Immunofluorescence (IFA) assays, western Blot (Western Blot) assays, and the like.
The invention discovers that aloe-emodin has lower cytotoxicity through in vitro cytotoxicity test, and half of cytotoxicity dose (CC 50 ) 15.5. Mu.M.
According to the invention, through in vitro cell experiments, aloe-emodin has remarkable functions of inhibiting the expression of ASFV structural protein p30 and inhibiting the replication of ASFV, and the inhibition effect is enhanced along with the increase of the drug concentration. Half-dose effective against ASFV (IC) in aloe-emodin in vitro cell assay 50 ) 1.1. Mu.M.
The invention provides an application of natural aloe-emodin or pharmaceutically acceptable salt thereof in preparing an anti-ASFV medicament and an application in preparing a medicament for preventing and treating African swine fever.
The invention provides an application of a pharmaceutical preparation prepared by taking aloe-emodin or pharmaceutically acceptable salt thereof as an effective ingredient and adding an auxiliary agent in resisting ASFV or preventing and treating African swine fever.
The pharmaceutical preparation is any veterinary clinically acceptable dosage form such as injection, powder, granule, tablet and the like.
The invention has the following advantages for preventing and treating African swine fever:
1. the aloe-emodin has stronger inhibitory activity on ASFV in vitro and also has remarkable ASFV-resisting activity at the concentration of 1.1 mu M, which indicates that the aloe-emodin has high-efficiency ASFV-resisting effect.
2. Aloe-emodin has low cytotoxicity and half cytotoxicity dose (CC in vitro cytotoxicity test 50 ) At 15.5 μm, a lower cytotoxic effect was only shown at more than 10 times higher than the effective concentration.
3. Aloe-emodin is a natural product widely existing in nature, and raw materials are widely available and low in cost; the preparation method is safe and reliable, and has great potential value in developing new drugs for preventing and treating African swine fever.
Drawings
FIG. 1 is a cytotoxicity assay of aloe-emodin on PAM cells using CCK-8 assay at various concentrations;
FIG. 2 is a graph showing the effect of qPCR on aloe-emodin on ASFV proliferation in PAM cells;
FIG. 3 is a method using an HAD 50 Determining the effect of aloe-emodin on ASFV titer in PAM cells;
FIG. 4 is a Western Blot analysis of the effect of aloe-emodin on ASFV p30 protein expression in PAM cells;
FIG. 5 is a graph showing the effect of aloe-emodin on ASFV p30 protein expression in PAM cells using IFA.
Detailed Description
In order to make the technical means, the creation characteristics, the purpose and the effects of the present invention easy to be clarified, the present invention is further described with reference to the following specific embodiments, but the scope of the present invention is not limited to the following embodiments.
The following experiments were all given biosafety and african swine fever laboratory activity permissions:
the animal biosafety third-level laboratory (ABSL-3) of China agricultural university has obtained the license of the agricultural rural department on developing African swine fever pathogen research and has been filed in the agricultural rural department, and meets the requirements of national biosafety level.
The reagents used in the experiments were all common commercial reagents unless otherwise specified; the methods of operation in the experiments are known in the art unless otherwise specified. The ASFV used in the following examples is a genotype II ASFV.
Example 1: cytotoxicity test of aloe-emodin with different concentrations on PAM cells by CCK-8 method
At 1.5×10 per well 5 PAM cells were inoculated into 96-well cell plates and cultured in a 37℃cell incubator, after the cells were completely adherent, aloe-emodin was diluted in a gradient with 1640 medium containing 2% serum, and added to the cells, with 3 multiple wells per aloe-emodin concentration. After 72h incubation in a 37℃cell incubator, the cell culture medium was discarded under light-protected conditions, and 100. Mu.L of serum-free 1640 medium and 10. Mu.L of CCK-8 solution (Biyunshan) were added to each well. After further incubation for 0.5h in the cell incubator, absorbance at a wavelength of 450nm was determined using a microplate reader. As can be seen from FIG. 1, aloe-emodin has a PAM cell viability of 50% at a concentration of 15. Mu.M.
Example 2: qPCR determination of the effect of aloe-emodin on ASFV proliferation in PAM cells
At 1.5×10 per well 5 PAM cells were inoculated into 96-well cell plates and cultured in a 37℃cell incubator, after the cells were completely adherent, aloe-emodin was diluted in gradient with 1640 medium containing 2% serum, aloe-emodin and virus (MOI=0.1) were added together into the cells, 3 duplicate wells were set for each aloe-emodin concentration, and placed in a 37℃incubator for culture. And (3) collecting a virus sample after 72h, repeatedly freezing and thawing for 3 times at-80 ℃, extracting a virus genome, detecting the copy number of the virus by qPCR, and calculating the inhibition ratio of aloe-emodin to the virus by using a primer in reference to the group standard 'African swine fever virus real-time fluorescence PCR detection method' T/CVMA 5-2018.
As shown in FIG. 2, aloe-emodin of the present invention has significant inhibitory effect on ASFV at concentrations ranging from 1.25 μm to 10. Mu.M, and exhibits dose dependence.
Example 3: half-red blood cell adsorption (HAD) 50 ) Determination of the Effect of aloe-emodin, a Natural product, on ASFV titres in PAM cells
At each hole 10 6 PAM cells were inoculated into 24-well cell plates and cultured in a 37℃cell incubator, after the cells were completely adherent, the natural product was diluted in a gradient with 1640 medium containing 2% serum, and a mixed solution of aloe-emodin and virus (MOI=0.1), which was a natural product, was added to the cells and placed in the 37℃incubator for culture. Collecting virus sample after 72 hr, repeatedly freezing and thawing at-80deg.C for 3 times, and diluting to 10 times -10 Dilutions were added to the cell plates, followed by 20 μl of 1% porcine red blood cell suspension per well. The cells were incubated in a 37℃incubator for 7 days, and the agglutination of the erythrocytes was observed daily. Calculation of the half-erythrocyte adsorption dose (HAD) of the virus 50 ) The method is a Reed-Muench two-step method.
As shown in FIG. 3, the aloe-emodin of the present invention has a remarkable inhibitory effect on the viral titer of ASFV infected with PAM cells at a concentration of 1.25 μm to 20. Mu.M, and exhibits remarkable dose dependence.
Example 4: western Blot analysis of the effect of aloe-emodin on ASFV p30 protein expression in PAM cells
At 5X 10 per well 6 PAM cells were inoculated into 6-well cell plates and cultured in a 37 ℃ cell incubator, after the cells were completely adherent, aloe-emodin was diluted in gradient with 1640 medium containing 2% serum, and a mixed solution of aloe-emodin and virus (moi=0.1) was added to the cells. Placing in a 37 ℃ incubator for culturing for 48 hours, discarding culture supernatant, washing with PBS for 2 times, adding 200 mu L of Western Blot cell lysate into each hole, ice-bathing for 15 minutes, collecting lysate, centrifuging at 4 ℃ for 15 minutes at 1200rpm, collecting supernatant, and carrying out Western Blot detection on ASFV p30 protein and beta-actin internal reference protein stripsA belt.
As shown in FIG. 4, the aloe-emodin of the present invention has a remarkable inhibitory effect on ASFV p30 protein expression of PAM-infected cells at a concentration of 2.5 μm-20. Mu.M, and exhibits remarkable dose dependence.
Example 5: IFA analysis of the effect of aloe-emodin on ASFV p30 protein expression in PAM cells
At each hole 10 6 PAM cells were inoculated into 24-well cell plates and cultured in a 37 ℃ cell incubator, after the cells were completely adherent, aloe-emodin was diluted in gradient with 1640 medium containing 2% serum, and a mixed solution of aloe-emodin and virus (moi=0.1) was added to the cells. After placing in a 37 ℃ incubator for culturing for 72 hours, the culture solution in the 96-well plate is discarded, and PBS solution is used for gently 1-2 times. 200 μl of 4% paraformaldehyde tissue fixative was added, the mixture was fixed at room temperature for 30min, the fixative was discarded, and the solution was washed 3 times with PBS for 5min each. 200. Mu.L of 0.1% Triton X-100 was added, left at room temperature for 20min, the solution was discarded, and washed 3 times with PBS solution. 200. Mu.L of 5% BSA solution was added, incubated at 37℃for 1h, the blocking solution was discarded and washed 3 times with PBS solution. ASFV p30 antibody was diluted 1:200, 200. Mu.L per well, incubated for 1h at 37℃and washed 3 times with PBS. FITC-labeled goat anti-pig fluorescent antibody was diluted to 5. Mu.g/mL, 200. Mu.L was added to each well, incubated at 37℃for 1h in the dark, and washed 3 times with PBS under the dark condition. Adding anti-fluorescence quenching sealing liquid, measuring fluorescence value by using a multifunctional enzyme label instrument, and observing fluorescence under a fluorescence microscope.
As shown in FIG. 5, the aloe-emodin of the present invention has a remarkable inhibitory effect on ASFV p30 protein expression of PAM-infected cells at a concentration of 1.25 μm-10. Mu.M, and exhibits remarkable dose dependence.
Claims (4)
1. Application of aloe-emodin or pharmaceutically acceptable salt thereof in preparing medicine for resisting African swine fever virus is provided.
2. Application of aloe-emodin or pharmaceutically acceptable salt thereof in preparing medicine for preventing and treating African swine fever is provided.
3. Use according to claim 1 or 2, characterized in that: the aloe-emodin or pharmaceutically acceptable salt thereof is used as an effective ingredient and auxiliary agents are used for preparing the pharmaceutical preparation.
4. A use according to claim 3, characterized in that: the pharmaceutical preparation comprises injection, powder, granule and tablet.
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