Application of polygonum capitatum or extract thereof in preparation of anti-pseudorabies virus medicine
By using water or alcohol extracts of Polygonum capitatum to prepare drugs against pseudorabies virus, the problem of the lack of effective drugs in the existing technology has been solved, and the drugs have achieved highly efficient inhibition and killing of pseudorabies virus, showing good application prospects.
Patent Information
- Application Number
- CN202511372578.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-24
- Publication Date
- 2025-11-07
AI Technical Summary
There is a lack of effective drugs against pseudorabies virus in the current technology. Synthetic chemical antiviral drugs have large toxic side effects and are prone to drug resistance. The protective efficacy of traditional vaccines has decreased, which poses a threat to the pig industry and public health security.
Aqueous or alcoholic extracts of Polygonum capitatum were used as anti-pseudorabies virus drugs. The extracts were prepared by decoction, maceration, percolation or ultrasonic extraction and combined with pharmaceutically acceptable carriers for the prevention and treatment of porcine epidemic diarrhea.
Polygonum capitatum extract can effectively kill viruses, block viral entry into cells, and inhibit viral replication, which is significantly better than the existing drug acyclovir. It has a strong inhibitory effect on pseudorabies virus, and the raw materials are abundant, low in cost, and simple in preparation process.
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Figure CN120899799A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and relates to application of polygonum capitatum or an extract thereof in preparation of an anti-pseudorabies virus drug. Polygonum capitatum The dry whole plant or aboveground part of polygonum capitatum Buch.-Ham. ex D.Don. BACKGROUND
[0002] Pseudorabies (PR) is an acute, severe, and highly contagious zoonosis caused by Pseudorabies virus (PRV). The host range is wide, and pigs are the only natural host. PRV infection can cause pregnant sows to abort and produce stillbirths, and the mortality rate of newborn piglets is as high as 100%. Adult pigs are latently infected and carry the virus for life, causing significant economic losses to the pig industry. In recent years, PRV variants have occurred frequently, and the protective efficacy of the traditional Bartha-K61 vaccine has decreased significantly. The protection effect on pigs is poor, and vaccinated herds can still be infected. In addition, since 2011, PR has broken out in many pig farms in China that have vaccinated with the vaccine, and the PRV wild virus antibody positive rate is high. The positive rate of pig farms is 75.75%-81.92%, and the positive rate of samples is 32.45%-36.03%. The current main epidemic variant in China belongs to the genetic type II, which is more virulent than the classic type I strain, causes more obvious clinical symptoms, spreads faster, and has a higher mortality rate. In recent years, research reports have shown that PRV can also infect humans and cause acute encephalitis and endophthalmitis in humans. The PRV hSD-1 / 2019 strain isolated from the cerebrospinal fluid of patients shows similar biological characteristics to the current epidemic PRV variant strain in pig herds in China. Therefore, pseudorabies not only seriously endangers the healthy development of the pig industry, but also poses a major threat to public health and safety due to the transmission of the virus across species. At present, there is no specific drug for pseudorabies in clinical practice, and synthetic chemical antiviral drugs have high toxicity and side effects, are prone to drug resistance, and have very limited clinical efficacy. Since 2005, China has completely banned the use of antiviral drugs such as amantadine, rimantadine, ribavirin, and acyclovir for food animals. Therefore, the development of anti-pseudorabies virus drugs is of great significance and practical value for the prevention and control of porcine epidemic diarrhea.
[0003] Polygonum capitatum is a polygonaceae plant Polygonum capitatumThe dried whole plant or above-ground part of Polygonum capitatum Buch.-Ham. ex D. Don has bitter, pungent and cool properties, and clearing heat and dampness, diuresis, relieving stranguria, activating blood and relieving pain effects. Polygonum capitatum is not only a commonly used traditional Chinese medicine in China, but also one of the characteristic Miao medicine resources in Guizhou Province, and is recorded in the Quality Standard of Chinese and National Medicine in Guizhou Province (2003 edition). Polygonum capitatum mainly contains flavonoids, phenolic acids, lignans, alkyl glycosides, organic acids, alcohols, esters, triterpenes, tannins and the like, and has antibacterial, anti-inflammatory, antipyretic, analgesic, anticancer, hypoglycemic, antioxidant and the like pharmacological activities. The single preparation of Polygonum capitatum, namely, Relinqing granules, has a unique effect on urinary system infection caused by Escherichia coli. However, so far, there is no report on the anti-pseudorabies virus effect of Polygonum capitatum.
[0004] The research of the present application shows that Polygonum capitatum and its extract can effectively directly kill viruses, block the entry of viruses into cells and inhibit the replication of viruses in cells, thereby playing an anti-pseudorabies virus role. SUMMARY
[0005] The primary object of the present application is to provide a new kind of medicine with an anti-pseudorabies virus effect, and specifically, the medicine comprises a water extract or an alcohol extract of Polygonum capitatum.
[0006] The second object of the present application is to provide a preparation method of the above-mentioned extract of Polygonum capitatum.
[0007] The further object of the present application is to provide a medicine composition as an anti-pseudorabies virus.
[0008] The still further object of the present application is to provide the use of Polygonum capitatum or the above-mentioned extract of Polygonum capitatum or the medicine composition in the preparation of a medicine for preventing and / or treating porcine epidemic diarrhea.
[0009] The Polygonum capitatum with an anti-pseudorabies virus effect of the present application is the whole plant of Polygonum capitatum Polygonum capitatum The dried whole plant or above-ground part of Polygonum capitatum Buch.-Ham. ex D. Don.
[0010] The preparation method of the extract of Polygonum capitatum of the present application comprises the following steps: a. adding Polygonum capitatum into a solvent for extraction; b. filtering the extract, combining the filtrates and concentrating; c. drying.
[0011] The extraction method in the above-mentioned step a is decoction, immersion, percolation, reflux or ultrasonic extraction.
[0012] The solvent in step a is ethanol aqueous solution with volume concentration of 0% to 100%; the solvent is used in an amount of 2 to 50 times of the medicinal material; the extraction temperature is 0 to 100℃; the extraction times is 1 to 5 times; and the extraction time is 0.5 to 5 hours each time.
[0013] The concentration temperature in step b is 25 to 100℃; and the relative density of the concentrated solution is 0.9 to 1.5.
[0014] The drying in step c includes normal pressure drying, reduced pressure drying, freeze drying or spray drying.
[0015] The pharmaceutical composition for preventing or treating pseudorabies virus of the present application contains a prophylactically or therapeutically effective amount of polygonum capitatum extract and a pharmaceutically acceptable carrier.
[0016] The effective amount of the polygonum capitatum extract of the present application is 0.01 g / kg to 10 g / kg body weight.
[0017] The polygonum capitatum extract and the pharmaceutical composition of the present application can be used for preventing and treating porcine epidemic diarrhea.
[0018] The pharmaceutically acceptable carrier as described above refers to the pharmaceutical carrier in the pharmaceutical field. For example, diluents, excipients such as water, physiological saline, glucose, mannitol, glycerol, ethanol and mixtures thereof; fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone, etc.; wetting agents such as glycerol, etc.; disintegrants such as calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds, etc.; surfactants such as Tween-80, etc.; lubricants such as talc, calcium stearate, magnesium stearate and polyethylene glycol, etc. In addition, other auxiliary materials such as flavoring agents, sweeteners, etc. can be added in the composition.
[0019] The pharmaceutical composition of the present application is administered to the subject in need of such prevention or treatment by oral, nasal inhalation, rectal, parenteral or transdermal administration.
[0020] The various dosage forms of the pharmaceutical composition of the present application can be prepared according to the conventional production method in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers, and then the desired dosage form is prepared.
[0021] The polygonum capitatum extract in the present application can effectively directly kill viruses, block the entry of viruses into cells and inhibit the replication of viruses in cells, and has a significant advantage over the antiviral drug acyclovir in inhibiting the proliferation of pseudorabies virus, and can be applied in the preparation of anti-pseudorabies virus drugs. The polygonum capitatum extract has abundant raw materials, low cost, simple preparation process and low cost, and has good development and application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 Effects of Polygonum capitatum extract on proliferation of porcine kidney PK15 cells, wherein, *** * indicates significant difference at 0.001 level compared with 0 µg / mL.
[0023] Figure 2 Effects of Polygonum capitatum extract on anti-pseudorabies virus, wherein, *** * indicates significant difference at 0.001 level compared with PRV group.
[0024] Figure 3 Effects of Polygonum capitatum extract on fluorescence intensity of porcine kidney PK15 cells infected with recombinant pseudorabies virus expressing green fluorescent protein.
[0025] Figure 4 Killing effects of Polygonum capitatum extract on pseudorabies virus, wherein, *** * indicates significant difference at 0.001 level compared with PRV group.
[0026] Figure 5 Blocking effects of Polygonum capitatum extract on adsorption of pseudorabies virus to porcine kidney PK15 cells, wherein, *** * indicates significant difference at 0.001 level compared with PRV group.
[0027] Figure 6 Inhibitory effects of Polygonum capitatum extract on replication of pseudorabies virus, wherein, *** * indicates significant difference at 0.001 level compared with PRV group. DETAILED DESCRIPTION
[0028] The present application is further illustrated by the following examples and figures, but not limited in scope.
[0029] Example 1: Preparation of Polygonum capitatum water extract Polygonum capitatum medicinal materials were taken, 10 times water was added, decocted twice, each time for 1.5 h. The decoction was filtered, the filtrate was combined, concentrated under reduced pressure to a relative density of 1.10-1.15 (60°C), filtered, and spray dried to obtain Polygonum capitatum water extract (yield 10.14%). Each gram of extract was equivalent to 9.86 g of raw medicinal materials in terms of gallic acid, 26.3 mg.
[0030] Example 2: Preparation of Polygonum capitatum alcohol extract Polygonum capitatum medicinal materials were taken, 8 times 75% ethanol was added, refluxed and extracted twice, each time for 2 h, filtered, the filtrate was combined, ethanol was recovered under reduced pressure until there was no alcohol smell, and continued to be concentrated, freeze-dried to obtain Polygonum capitatum alcohol extract.
[0031] Example 3: Effect of Polygonum capitatum extract on the proliferation of porcine kidney PK15 cells 0.75×10 5 A suspension of porcine kidney PK15 cells per mL was seeded at 100 μL per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h until a monolayer formed. 100 μL of DMEM culture medium or diluted PCEP (Polygonum capitatum aqueous extract) at different concentrations (25, 50, 100, 200, and 400 μg / mL) was added to each well, and the plates were incubated at 37°C with 5% CO2 for 2 h and 6 h, respectively. The supernatant was discarded, and 100 μL of DMEM culture medium containing 2% fetal bovine serum was added to each well. The plates were incubated at 37°C with 5% CO2 for 46 h and 42 h, respectively. Four h 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 490 nm using a microplate reader.
[0032] The results are as follows Figure 1 As shown, concentrations of Polygonum capitatum extract (PCEP) greater than 100 µg / mL significantly inhibited the proliferation of porcine kidney PK15 cells. P The concentration of Polygonum capitatum extract (< 0.001) against PK15 cells was 100 µg / mL, with a maximum safe concentration (MNTC) of 100 µg / mL. Therefore, 100 µg / mL was chosen as the initial concentration for the experiment on the anti-pseudorabies virus activity of Polygonum capitatum extract.
[0033] Example 4: Anti-pseudorabies virus effect of Polygonum capitatum extract Take a concentration of 0.75 × 10 5 PK15 cell suspension at 100 μL / well was seeded into 96-well plates and incubated at 37°C, 5% CO2 for 24 h until a monolayer formed. DMEM culture medium, different concentrations of Polygonum capitatum extract dilution (6.25, 25, 100 μg / mL), or acyclovir (200 μM) were added to each well, and the plates were incubated at 37°C, 5% CO2 for 2 h. The supernatant was discarded, and DMEM culture medium or 200 TCID45 solution was added to each well. 50Pseudorabies virus diluent 50 μL, then add DMEM culture solution or different concentrations of polygonum capitatum extract diluent (12.5, 50, 200 μg / mL) or acyclovir (400 μM) 50 μL, place in a 37°C, 5% CO2 incubator for culture, shake every 30 min. Incubate for 2 h, discard the virus and drug mixture in the well, add DMEM culture solution or different concentrations of polygonum capitatum extract diluent (6.25, 25, 100 μg / mL) or acyclovir (200 μM) 100 μL, place in a 37°C, 5% CO2 incubator for incubation for 2 h. Discard the supernatant, add maintenance solution (DMEM culture solution containing 2% fetal bovine serum) 100 μL per well, place in a 37°C, 5% CO2 incubator for incubation for 42 h. 4 h before the end of the culture, add 2 mg / mL MTT solution 50 μL per well, shake on a shaker, and wait for the crystals to completely dissolve. Measure the OD value at a wavelength of 490 nm using a microplate reader. Calculate the inhibition rate of pseudorabies virus according to the following formula.
[0034] Inhibitory rate (%) = (test group OD value - virus control group OD value) / (cell control group OD value - virus control group OD value) x 100%.
[0035] The results are shown in Figure 2 polygonum capitatum extract (PCEP) can significantly inhibit the proliferation of pseudorabies virus (PRV) in PK15 cells, and show a concentration-dependent relationship. The inhibition rates of polygonum capitatum extract (PCEP) at concentrations of 50 and 100 μg / mL on the proliferation of PRV in PK15 cells were 77.70% and 96.12%, respectively, which were significantly better than the positive control drug acyclovir (ACV), which had an inhibition rate of only 43.78% on the proliferation of PRV in PK15 cells.
[0036] Example 5: Effect of polygonum capitatum extract on the fluorescence intensity of PK15 cells infected with green fluorescent recombinant pseudorabies virus The strain used was a green fluorescent protein-expressing pseudorabies virus (rPRV-GFP). Take the PK15 cells cultured for 24 h in a 24-well plate, set up cell control group, virus control group, acyclovir (ACV) control group and polygonum capitatum extract test group, and treat the cells with drugs and viruses according to the method of Example 4. Discard the culture solution, add 300 μL of 1x Hoechst 33342 live cell staining solution per well, and place in an incubator for 10 min. Discard the culture solution, add phosphate buffer solution (PBS), and observe under a fluorescence microscope and take photographs.
[0037] The results are shown in Figure 3As shown, the green fluorescence intensity of PK15 cells infected with pseudorabies virus expressing green fluorescent protein (rPRV-GFP) was reduced in a concentration-dependent manner by PCEP, and the inhibitory effect of PCEP at concentrations of 50 and 100 μg / mL was superior to that of the positive control drug acyclovir (ACV), further confirming that PCEP has an anti-pseudorabies virus (PRV) effect.
[0038] Example 6: Killing effect of PCEP on pseudorabies virus DMEM culture solution or different concentrations of PCEP dilutions (12.5, 50, 200 μg / mL) or acyclovir (400 μM) were mixed with 200 TCID 50 The pseudorabies virus dilution was mixed with an equal volume of PCEP, and incubated at 37°C in a 5% CO2 incubator for 2 h. 100 μL was added to a 96-well plate of PK15 cells that had been adhered and grown into a monolayer for 24 h, with 4 replicate wells. At the same time, blank controls, normal cell controls, and virus controls were set up. Incubation was carried out at 37°C in a 5% CO2 incubator, with shaking every 30 min. After 2 h of incubation, the virus and drug mixture in the wells was discarded, and 100 μL of maintenance solution (DMEM culture solution containing 2% fetal bovine serum) was added to each well, and incubation was carried out at 37°C in a 5% CO2 incubator for 70 h. Cell pathology (CPE) was observed every day. 50 μL of 2 mg / mL MTT solution was added to each well 4 h before the end of the incubation, and the shaking instrument was shaken until the crystals were completely dissolved. The OD value was measured at a wavelength of 490 nm using a microplate reader, and the killing rate of pseudorabies virus was calculated according to the following formula.
[0039] Killing rate (%) = (OD value of the test group - OD value of the virus control group) / (OD value of the cell control group - OD value of the virus control group) x 100%.
[0040] The results are shown in Table 2. Figure 4 As shown, PCEP had a significant killing effect on PRV, and the effect was concentration-dependent, with the killing rates of PCEP at concentrations of 25 and 100 μg / mL being 77.36% and 98.06%, respectively, which were significantly higher than that of the positive control drug acyclovir (45.40%).
[0041] Example 7: Blocking effect of PCEP on the adsorption of pseudorabies virus to PK15 cells A concentration of 0.75 x 10 5PK15 cell suspension at 100 μL / well was seeded into 96-well plates and incubated at 37°C, 5% CO2 for 24 h until a monolayer formed. 100 μL of DMEM culture medium, or different concentrations of Polygonum capitatum extract dilution (6.25, 25, 100 μg / mL), or acyclovir dilution (200 μM) was added to each well, and the plates were incubated at 37°C, 5% CO2 for 2 h. The supernatant was discarded, and 100 μL of DMEM culture medium or 100 TCID45 solution was added to each well. 50 100 μL of pseudorabies virus dilution buffer 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 maintenance medium (DMEM culture medium containing 2% fetal bovine serum) 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 culture 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. The OD value was measured at 490 nm using a microplate reader. The blocking rate of pseudorabies virus adsorption on PK15 cells was calculated using the following formula.
[0042] 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%.
[0043] The results are as follows Figure 5 As shown, Polygonum capitatum extract (PCEP) significantly blocked pseudorabies virus (PRV) entry into PK15 cells in a concentration-dependent manner. Specifically, PCEP at a concentration of 200 μg / mL showed a PRV blocking rate of 66.84%, significantly higher than the positive control drug acyclovir (34.43%).
[0044] Example 8: Inhibitory effect of Polygonum capitatum extract on pseudorabies virus replication Take a concentration of 0.75 × 10 5 PK15 cell suspension at 100 μL / well was seeded into 96-well plates and incubated at 37°C in a 5% CO2 cell culture incubator for 24 h until a monolayer was formed. The supernatant was discarded, and DMEM or 100 TCID50 was added to each well. 50100 μL of each pseudorabies virus dilution buffer was added and incubated in a 37°C, 5% CO2 incubator, with shaking every 30 min. After 2 h of incubation, the virus solution in the wells was discarded. 100 μL of DMEM culture medium, or different concentrations of Polygonum capitatum extract dilution (6.25, 25, 100 μg / mL), or acyclovir dilution (200 μM) 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 maintenance medium (DMEM culture medium containing 2% fetal bovine serum) 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 culture, 50 μL of 2 mg / mL MTT solution was added to each well, and the wells were shaken until the crystals were completely dissolved. The OD value was measured at 490 nm using a microplate reader, and the inhibition rate of pseudorabies virus replication in PK cells was calculated using the following formula.
[0045] 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%.
[0046] The results are as follows Figure 6 As shown, Polygonum capitatum extract (PCEP) significantly inhibited pseudorabies virus (PRV) replication in PK15 cells in a concentration-dependent manner. Specifically, PCEP at a concentration of 100 μg / mL inhibited PRV replication by 33.29%, significantly higher than the positive control drug acyclovir (22.73%).
[0047] In summary, the Polygonum capitatum extract of this invention can effectively and directly kill viruses, block viral entry into cells, and inhibit viral replication within cells, thus exerting an anti-pseudorabies virus effect. Its inhibitory effect on pseudorabies virus proliferation is significantly superior to that of the antiviral chemical drug acyclovir, and it can be used in the preparation of anti-pseudorabies virus drugs. Polygonum capitatum extract is an abundant and inexpensive raw material, with a simple preparation process and low cost, showing promising development and application prospects.
Claims
1. Use of Polygonum capitatum or an extract thereof in the preparation of a medicine for the treatment of pseudorabies virus, characterized in that, The headleaf smartweed is Polygonaceae plant headleaf smartweed Polygonum capitatum Dry whole plant or aerial part of Humulus scandens (Lour.) Merr.
2. The use of the polygonum capitatum or the extract thereof according to claim 1 in the preparation of a medicine for resisting the pseudorabies virus, characterized in that, The preparation method of the Polygonum capitatum extract comprises: a. adding Polygonum capitatum into a solvent for extraction; b. filtering the extract, combining the filtrates, and concentrating; c. drying.
3. Use according to claim 2, characterized in that, The extraction method is decocting, soaking, percolation, refluxing, or ultrasonic extraction.
4. Use according to claim 2, characterized in that, The solvent is 0%-100% ethanol aqueous solution, and the mass amount is 2-50 times of the amount of Polygonum capitatum medicinal material.
5. Use according to claim 2, characterized in that, The extraction temperature is 0-100 ℃, the extraction times are 1-5 times, and the extraction time is 0.5-5 hours each time.
6. Use according to claim 2, characterized in that, The concentration temperature is 25-100 ℃, and the relative density of the concentrated solution is 0.9-1.
5.
7. An anti-pseudorabies virus drug, characterized by, Polygonum capitatum or its extract is used as the active ingredient.
8. The medicament according to claim 7, wherein The medicine also comprises a pharmaceutically acceptable carrier.