Traditional Chinese medicine composition for preventing African swine fever attenuated virus infection, medicine prepared from composition and corresponding preparation method

Abstract

The invention belongs to the field of veterinary drugs, and particularly relates to a traditional Chinese medicine composition for preventing African swine fever attenuated virus infection, a medicine prepared from the composition and a corresponding preparation method. The traditional Chinese medicine composition comprises the following components in parts by weight: 20-40 parts of rhizoma curculiginis, 5-15 parts of cynanchum thesioides, 5-15 parts of longspurred fairybells rhizome, 5-15 parts of triquetrous tadehagi root, 5-15 parts of apium graveolens, 10-20 parts of myricaria laxiflora and 10-20 parts of carex The traditional Chinese medicine composition is a pure traditional Chinese medicine preparation, is safe, free of toxic and side effects and palatability, has the characteristics of strengthening the body resistance to eliminate pathogenic factors, exact in curative effect, green, safe and capable of being mixed with feed / drinking water for use, can obviously reduce the morbidity of African swine fever and improve the health degree of swinery, and has good market application and popularization values.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary medicine, and specifically relates to a traditional Chinese medicine composition for preventing African swine fever attenuated virus infection, a medicine prepared from the composition and a corresponding preparation method. Background Art

[0002] Under the current situation of normalized prevention and control of African swine fever, the emergence of weak strains of African swine fever virus (ASFV) has brought considerable difficulties to the biosafety prevention and control of pig farms, and the survival of pig farms is facing severe challenges. Different from the previous strong strains of gene type II, which have the characteristics of fast onset, acute onset, and high mortality, after the pigs are infected with weak strains of African swine fever, they have strong horizontal transmission ability, long incubation period, low level of toxin carrying and excretion, long and irregular duration, strong clinical concealment, difficult early monitoring, and great impact on the production performance of sow groups. Usually, sows are the first to get sick. In the early stage of the diseased pigs, the main manifestation is a decrease in feed intake, and other clinical symptoms are not obvious; in the later stage, repeated high fever of about 41°C will not subside, appetite will be significantly reduced or even abolished, mental state will be poor, and they are unwilling to move, but the sick pigs have no respiratory symptoms, vomiting, or bloody stools. Some pigs can last for one to two weeks or even longer without dying.

[0003] The clinical symptoms after attenuated virus infection are milder than those of virulent virus infection, and are mainly characterized by subacute and chronic. Subacute: The clinical symptoms are relatively mild, mainly mild fever, loss of appetite, depression, abortion in pregnant sows, etc., or even no obvious clinical symptoms, and the course of the disease is generally between 5 and 30 days. Death occurs within 15 to 45 days after infection, and the mortality rate is low and varies greatly in different cases (such as 10% to 70%). Chronic: The clinical manifestations are diverse, including weight loss, intermittent fever, respiratory symptoms, skin necrosis, chronic skin ulcers, arthritis, etc., and the anatomical pathology shows pericarditis, pulmonary adhesions, and joint effusions. The course of the disease is long, lasting 2 to 15 months, or even longer, and the mortality rate is low. Because the clinical symptoms of non-swine fever attenuated virus are relatively mild, it is not easy for on-site personnel to detect it. In addition, if the pig farm has underlying diseases such as blue ear disease, pseudorabies, and classical swine fever, the concealment of non-swine fever attenuated virus is further increased. When clinically found to be infected with non-swine fever attenuated virus, the virus has often existed in the pig farm for some time, and the infection rate is already high.

[0004] The clinical symptoms of infection with weak strains of African swine fever virus are atypical and vary greatly from individual to individual. Pregnant sows show symptoms such as repeated fever and anorexia, some sows have miscarriages, a high proportion of stillbirths, scabs on the breasts, and poor vitality of newborn piglets. Under non-stress conditions in fattening groups, the symptoms are not obvious, and under stress conditions, symptoms such as skin necrotic spots, enlarged joints and spleen, and necrosis are shown. It brings great difficulties to front-line clinical production and disease prevention and control. In view of the hazards of weak strains, the Ministry of Agriculture and Rural Affairs issued the "Technical Guidelines for the Prevention and Control of Weak Strains of African Swine Fever Virus in Farms" in November 2023, providing guidance from multiple angles such as disease monitoring and biosafety. However, the industry still lacks effective drugs for weak strains to increase the survival rate of infected pigs, reduce economic losses, and prevent the spread of the virus. In the current situation where vaccine research and development is in a dilemma, the development of preventive drugs to protect susceptible animals from the invasion of weak strains of African swine fever is of great practical significance for the prevention and control of ASFV.

[0005] A large amount of literature and clinical verification have confirmed that the active ingredients of traditional Chinese medicine such as flavonoids, polyphenols, polysaccharides, saponins, etc. can stimulate the body's nonspecific immune function after being absorbed by the gastrointestinal tract, and increase the body's infection threshold by expressing interferon and antibodies, protecting susceptible animals from infection by current weak strains, and have great research and development value for improving the survival rate and breeding efficiency of farms. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a traditional Chinese medicine composition for preventing African swine fever attenuated infection, and also provide a medicine prepared from the composition and a corresponding preparation method.

[0007] Based on the above purpose, the present invention adopts the following technical solution: The first aspect of the present invention provides a traditional Chinese medicine composition for preventing attenuated African swine fever infection, wherein the weight composition of the traditional Chinese medicine composition is: 20-40 parts of white curculigo, 5-15 parts of radix ophiopogonis, 5-15 parts of dogtail ginseng, 5-15 parts of cucurbitacin root, 5-15 parts of celery, 10-20 parts of water cypress branches, and 10-20 parts of cruciferous wormwood.

[0008] Preferably, the weight composition of the traditional Chinese medicine composition is: 30 parts of white curculigo, 10 parts of radix scutellariae, 10 parts of dogtail ginseng, 10 parts of cucurbitacin root, 10 parts of celery, 15 parts of water cypress branches, and 15 parts of cruciferous wormwood.

[0009] The second aspect of the present invention provides a drug for preventing African swine fever attenuated virus infection, which is prepared from an extract of the traditional Chinese medicine composition described in the first aspect and pharmaceutically acceptable excipients.

[0010] Preferably, the dosage form of the drug is an oral dosage form.

[0011] Preferably, the medicine is in the form of granules, oral solution or extract.

[0012] Preferably, the extract is an aqueous extract of the traditional Chinese medicine composition.

[0013] The third aspect of the present invention provides a method for preparing the medicine described in the second aspect, comprising the steps of extracting the extract: mixing the medicinal ingredients of the traditional Chinese medicine composition according to weight proportions, subjecting the mixture to steam explosion treatment, adding water for decoction, filtering, and collecting the filtrate to obtain the extract of the traditional Chinese medicine composition.

[0014] Preferably, the steam explosion treatment process has a steam pressure of 3.5 to 6 MPa and a treatment time of 4 to 8 min.

[0015] Preferably, the filtration is carried out using a filter press device with a pressure of 0.10-0.12 MPa and a filter cloth pore size of 1000-1200 mesh.

[0016] Preferably, the drug is a granule, and the preparation steps include: concentrating the extract at 55-60° C. to prepare an extract, adding auxiliary materials, granulating, and sieving to obtain the granule.

[0017] Preferably, the auxiliary material is any one of maltodextrin and glucose.

[0018] Preferably, an MVR evaporator is used to concentrate the extract at a concentration temperature of 55-60°C.

[0019] Preferably, the concentrated extract is further concentrated using a spherical condenser until a paste having a relative density of 1.15 to 1.2 at 55 to 60° C. is formed.

[0020] Preferably, the granulation is carried out by a fluidized bed drying granulator with an air inlet temperature of 65-70°C.

[0021] Preferably, the method of using the granules is: mix 1 kg of granules with 500 kg of feed, and feed freely for 15 to 30 days.

[0022] All components in the Chinese medicine composition of the present invention can be purchased from the market. The formulation principle of the Chinese medicine composition is as follows: White Curculigo is the root of the plant Sea Cucumber of the family Dipsacus. It is harvested in autumn, washed, and used fresh or sliced ​​and dried. It tastes sweet and slightly bitter, and is warm in nature. It benefits the lungs and spleen, strengthens the kidneys and yang, and promotes blood circulation and relaxes muscles. It is used to treat cough due to lung deficiency, indigestion due to spleen deficiency, impotence due to kidney deficiency, leucorrhea, and uterine prolapse.

[0023] The ground melon is the whole herb of the plant Glechoma tenuifolia of the Apocynaceae family. It tastes sweet and is cool in nature. It enters the lung meridian. It replenishes lung qi, clears away heat and reduces fire, promotes the production of body fluids and quenches thirst, and relieves inflammation and pain. It is used to treat virtual fire inflammation, sore throat, insufficient qi and yin, fatigue and forgetfulness, restlessness and thirst, dizziness and insomnia, postpartum weakness, and insufficient milk.

[0024] Dogtail ginseng is the rhizome or root of the plant of the Liliaceae family. It tastes sweet and is neutral in nature. It replenishes qi and yin and moistens the lungs. It treats lung dryness due to qi and yin deficiency, bone steaming and tidal fever, soreness of the waist and knees, and night sweats.

[0025] Gourd tea root is the root of Gourd tea and Gourd tea of ​​Leguminosae. It tastes slightly bitter and pungent, and is neutral in nature. It enters the lung and liver meridians, clears away heat and relieves cough, removes toxins and disperses stagnation. It is used to treat wind-heat cough, lung abscess, carbuncle, scrofula and jaundice.

[0026] Celery is the dried mature fruit of the Umbelliferae plant. It is harvested in late summer and early autumn when the fruit is ripe and dried in the sun. It is slightly sour, cool and spicy. It clears lung heat, increases stomach fire and helps digestion. It is used in Tibetan medicine for "Bacon" disease, lung heat syndrome, stomach cold and abdominal distension, and indigestion.

[0027] Water cypress branch, this product is the dried young branches of the plant water cypress branch of the Tamarix family and several plants of the same genus. Collected in spring and summer, dried in the sun. Astringent, sweet, and cool. Clears away heat and detoxifies, dispels and clears rashes. Used for measles that does not clear up, sore throat, poisoning, yellow water disease, blood heat disease, plague epidemic, and visceral toxic heat.

[0028] Cross Carex is the whole herb of Cross Carex of the Cyperaceae family. It is harvested in summer and autumn, washed, cut into sections and dried. It tastes spicy and sweet and is neutral in nature. It can relieve the symptoms of the disease and clear rashes; regulate qi and strengthen the spleen. It is used to treat wind-heat colds; measles that are not clear; and indigestion.

[0029] Since the clinical symptoms of the attenuated African swine fever virus are relatively mild, the incubation period is long, and there is even intermittent detoxification, there are often no symptoms when infection occurs. In essence, the incubation period and the asymptomatic infection period are actually overlapping stages. From the perspective of macroscopic pathogenesis of traditional Chinese medicine, even if there are microscopic laboratory indicators showing positive viral nucleic acid, before the onset of symptoms, asymptomatic infected persons are still in the "lying but not yet onset" stage of febrile disease transmission. Therefore, the time when the pathogen is hidden, that is, the onset time window, refers to the incubation period and the asymptomatic period after infection of some patients. The asymptomatic period may persist, or symptoms may gradually appear.

[0030] Chinese veterinary medicine believes that the cause of latent evil (such as weak virus infection) invading pigs is insufficient positive energy and strong evil energy. Just as "Suwen·Acupuncture Theory" believes that "positive energy exists inside, evil energy cannot interfere", which means that positive energy can overcome evil energy, and as long as positive energy is sufficient, the disease will not occur. "Suwen·Comment on Fever Theory" says: "Where evil energy gathers, its energy must be weak", which means that insufficient positive energy is the internal cause of the body's infection with evil energy. At the same time, when evil energy invades the pig's body, if positive energy overcomes evil energy, the disease will not occur, but if evil energy overcomes positive energy, the disease will occur. Preventing and treating epidemic diseases based on the theory of latent evil energy is helpful to identify the key points of symptoms, formulate core treatment methods for the pathogenesis, cut off the disease, and truly achieve the purpose of preventing infection and disease. He Lianchen, a famous doctor in the Qing Dynasty, pointed out: "Doctors must know latent energy, so as not to see the disease and treat the disease, and can grasp the opportunity before the disease symptoms appear"; Wu Youke, an infectious disease expert, proposed that plague is "evil energy hiding in the membrane source", and the treatment advocates "clearing the membrane source and reversing the disease site" and "early removal of the root cause of the disease is the key". Both doctors emphasized that the hidden evil should be the core of the treatment of the epidemic. The evil must be expelled early and directly to the site of the disease, so as to develop effective prevention and treatment prescriptions without being disturbed by complex and concurrent symptoms.

[0031] The present invention analyzes the pathogenesis of the weak strain and verifies the prescriptions for multiple clinical screenings. It is believed that the prevention of the weak strain of African swine fever meets the characteristics of strong evil and weak body, weak exterior and weak body, insufficient lung qi, qi deficiency and toxicity, and hidden evil. The treatment is suitable for invigorating qi and tonifying the lung, detoxifying and clearing rash. In the prescription, white curculigo is reused as the main medicine to strengthen the spleen and benefit the lung, nourish the lung qi, and enhance the function of protecting the throat and the skin; the ground cucumber is used to nourish the lung and produce fluid, the dogtail ginseng moisturizes and nourishes yin, the gourd tea root clears the lung and removes toxins, and the fragrant celery clears the heat of the lung and stomach, which are all ministerial medicines, assisting the main medicine to clear stomach fire and moisten the lung fluid; water cypress branches and cross moss clear heat and clear rash as adjuvants, assisting the main medicine to eliminate evil and strengthen the body, and all the medicines are used together to play the functions of invigorating qi and strengthening the exterior, strengthening the spleen and clearing the lung, so that the surface deficiency is solid, the lung qi is full, the hidden fire is cleared, and the throat is protected, and the body is strengthened and the evil is eliminated, and a two-pronged approach is taken to improve the health of the pig herd and reduce the risk of African swine fever weak virus infection.

[0032] Compared with the prior art, the present invention has the following beneficial effects: (1) The Chinese medicine composition of the present invention has a significant effect on reducing the expression of ASFV-p72 gene and has a good effect on inhibiting viral replication. In clinical trials, the present invention can effectively reduce the incidence of clinical infection of African swine fever attenuated strains.

[0033] (2) The present invention is a pure Chinese medicine preparation, which is safe, has no toxic side effects, and has no palatability problems. It has the characteristics of strengthening the body and eliminating evil, has a definite therapeutic effect, is green and safe, and can be mixed with feed / drinking water. It can significantly reduce the incidence of African swine fever and improve the health of pigs, and has good market application and promotion value. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail by way of embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] Example 1 The present embodiment provides a traditional Chinese medicine composition for preventing attenuated African swine fever infection, wherein the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 30 parts of white curculigo, 10 parts of radix ophiopogonis, 10 parts of dogtail ginseng, 10 parts of cucurbitacin root, 10 parts of celery, 15 parts of water cypress branches, and 15 parts of cruciferous wormwood.

[0036] The preparation method of the above-mentioned Chinese medicine composition comprises the following steps: (1) Mix the APIs according to their weight proportions, load them into a steam explosion device, introduce saturated steam, maintain the steam pressure at 3.5 MPa for 8 min, then open the pressure relief valve to perform steam explosion to obtain API powder; (2) The raw material drug powder after steam explosion is boiled and extracted with water, and the extracted drug liquid and drug residue are transferred to the filter press device for filtration. The pressure is controlled at 0.12 MPa, and the filter cloth pore size is 1000 mesh. After the filtration is completed, the filtrate is collected; (3) The filtrate was transferred to an MVR evaporator for concentration at a concentration temperature of 60°C. A spherical condenser was used to further concentrate the concentrated filtrate until an extract with a relative density of 1.15 at 60°C was formed. Then, 80 parts of auxiliary material maltodextrin was added and granulation was performed in one step using a boiling drying granulator. The inlet air temperature was controlled at 65°C during granulation. After granulation, the whole granules were sieved and packaged to obtain granules.

[0037] Example 2 The present embodiment provides a traditional Chinese medicine composition for preventing attenuated African swine fever infection, wherein the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 20 parts of white curculigo, 15 parts of radix ophiopogonis, 15 parts of dogtail ginseng, 15 parts of cucurbitacin root, 15 parts of celery, 10 parts of water cypress branches, and 10 parts of cruciferous wormwood.

[0038] The preparation method of the above-mentioned Chinese medicine composition comprises the following steps: (1) Mix the APIs according to their weight proportions, load them into a steam explosion device, introduce saturated steam, maintain the steam pressure at 6.0 MPa for 4 min, then open the pressure relief valve to perform steam explosion to obtain API powder; (2) The raw material drug powder after steam explosion is boiled and extracted with water, and the extracted drug liquid and drug residue are transferred to the filter press device for filtration. The pressure is controlled at 0.11MPa, and the filter cloth pore size is 1100 mesh. After the filtration is completed, the filtrate is collected; (3) The filtrate was transferred to an MVR evaporator for concentration at a concentration temperature of 55°C. A spherical condenser was used to further concentrate the concentrated filtrate until a paste with a relative density of 1.15 at 55°C was formed. Then, 90 parts of auxiliary material maltodextrin was added and granulation was performed in one step using a boiling drying granulator. The inlet air temperature was controlled at 70°C during granulation. After granulation, the whole granules were sieved and packaged to obtain granules.

[0039] Example 3 The present embodiment provides a traditional Chinese medicine composition for preventing attenuated African swine fever infection, wherein the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 40 parts of white curculigo, 5 parts of radix ophiopogonis, 5 parts of panax notoginseng, 5 parts of radix cucurbitae, 5 parts of celery, 20 parts of water cypress branches, and 20 parts of carex crossifolia.

[0040] The preparation method of the above-mentioned Chinese medicine composition comprises the following steps: (1) Mix the APIs according to their weight proportions, load them into a steam explosion device, introduce saturated steam, maintain the steam pressure at 4.5 MPa for 6 minutes, then open the pressure relief valve to perform steam explosion to obtain API powder; (2) The raw material drug powder after steam explosion is boiled and extracted with water, and the extracted drug liquid and drug residue are transferred to the filter press device for filtration. The pressure is controlled at 0.10 MPa, and the filter cloth pore size is 1200 mesh. After the filtration is completed, the filtrate is collected; (3) The filtrate is transferred to an MVR evaporator for concentration at a concentration temperature of 58°C; a spherical condenser is used to further concentrate the concentrated filtrate until a paste with a relative density of 1.2 at 60°C is formed, and then 80 parts of auxiliary material maltodextrin is added. A boiling drying granulator is used for one-step granulation. The inlet air temperature during granulation is controlled at 68°C. After granulation, the whole granules are sieved and packaged to obtain granules.

[0041] Example 4 In vitro inhibition test 1. Test materials (1) Cell line: The primary PAM cell line was derived from 8 kg of bronchoalveolar lavage fluid from SPF weaned piglets.

[0042] (2) Virus fluid: The attenuated strain of African swine fever virus (SD / DY-I / 21) was isolated and preserved by the African swine fever regional laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0043] (3) Materials and reagents: Australian fetal bovine serum was purchased from Sigma-Aldich; 96-well cell plates were purchased from Thermo Fisher Scientific; RPMI-1640 medium was purchased from Gibco; cell proliferation-toxicity detection kit was purchased from Dalian Meilun Biotechnology Co., Ltd.; 0.22 μm microporous filter membrane and dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher Scientific; total RNA rapid extraction kit and penicillin-streptomycin (double antibody) were purchased from Shanghai Shangbao Biotechnology Co., Ltd.; HiScript III 1stStrand cDNA Synthesis Kit (+gDNAwiper) and ChamQ SYBR qPCR Master Mix were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0044] 2. Test methods (1) Isolation, culture and counting of primary PAM cells: Weaned piglets were anesthetized, and the axillary vein was cut with a scalpel to bleed and kill. The chest cavity was opened, and the lungs were removed as a whole and placed in PBS containing 5% double antibody. The lungs were observed to be intact and quickly transferred to the cell station. The lungs were washed several times with PBS containing 5% double antibody, and the hemostatic forceps were removed. The lung lobules were wrapped with gauze, and 5% double antibody-containing PBS was added to the bronchi for lavage. 400 mL of PBS was added each time, and lavage was performed 4 times in total. The lavage fluid was collected into a 50 mL centrifuge tube and centrifuged at 300 g for 10 min. The supernatant was discarded and the precipitate was blown with 1640 containing 5% double antibody. 1 mL was added to each tube and collected into a 50 mL centrifuge tube. The supernatant was discarded and the precipitate was added with an appropriate amount of freezing solution. The cells were counted with trypan blue. The experiment showed that the obtained primary porcine PAM cells were in good condition. The trypan blue counting method showed that the PAM cell density was about 6*10 6 pcs / mL. Dispense 1 mL of each tube into cryopreservation tubes for cryopreservation.

[0045] (2) Preparation of Chinese medicine samples: According to the raw material proportions and preparation methods of Examples 1 to 3, 1000 g of Chinese medicine was weighed to make 1000 g of granules. After high-pressure steam sterilization (121.3°C, 15 min), a 1 g / L solution was prepared using phosphate buffered saline (PBS) as solvent, labeled and stored at 4°C for future use.

[0046] (3) Virus TCID50 determination: Take 1 mL of subcultured PAM cells from a liquid nitrogen tank, bathe in 37°C water until dissolved, centrifuge at 500 g for 5 min, remove the supernatant, dilute to 24 mL with 1640 medium containing 2% double antibody and 20% serum, let it adhere to the wall overnight, gently aspirate and discard the cell culture medium in the 96-well plate, add 100 μL PBS and gently wash 2 to 3 times, add the above-mentioned 1640 medium without serum, without double antibody, and containing virus, 100 μL per well (set 6 to 8 different concentration gradients), 8 replicates for each concentration, and set up a blank control group. After the virus is incubated at 37°C for 2 h, gently discard the virus solution, add 1640 medium without double antibody and containing 10% serum, 100 μL per well, continue incubation for 24 h, then add 20 μL porcine red blood cells, culture for 24 h, observe and record the hemagglutination phenomenon, calculate TCID50, and the final measured TCID50 titer = 10 5.6 The test attack dose was determined to be 10 3 TCID50.

[0047] (4) Determination of safe concentration of traditional Chinese medicine: PAM cells were taken out of liquid nitrogen and inoculated into 96-well plates after recovery, with 100 μL per well, leaving a single blank control, and incubated at 37°C and 5% CO 2 The drug solution prepared in (2) was diluted 1640 times to 10 different concentration gradients (2 -1 , 2 -2 ……2 -10 ). After 24 hours of culture, the culture medium in the cell plate was aspirated, and 100 μL of the diluted drug solution was added to the cell plate, which was divided into the experimental group and the control group. 100 μL of 1640 was added to the experimental group and the control group respectively. After 48 hours, the liquid in each well was aspirated, and 100 μL of 10% CCK was added to continue incubation for 1 to 4 hours. The absorbance value was measured at 450nm using an ELISA reader. The cell survival rate was calculated, and finally the safe concentration of the three drug test groups for PAM cells was measured to be 7.8 to 15.6 mg / mL.

[0048] (5) Detection of African swine fever virus-P72 gene expression in PAM cells by qRT-PCR: 1) Extract total RNA from samples: Use a spin column animal RNA extraction kit to extract total RNA from samples.

[0049] 2) Reverse transcription to synthesize cDNA: Remove genomic DNA from RNA samples: Prepare a reaction mixture and add it to an RNase-free centrifuge tube. Mix it by gently blowing. Incubate it in a 42℃ water bath for 2 minutes. Prepare the reverse transcription reaction system. Mix it by gently blowing. Incubate it at 37℃ for 15 minutes for reverse transcription reaction, and then incubate it at 85℃ for 5 seconds. The obtained cDNA is packaged and stored at -80℃.

[0050] 3) Primer design: Based on the target gene mRNA sequence obtained from the NCBI gene library (GenBank: MZ945537), the universal primer P72-U (5 , -GGCACAAGTTCGGACATGT-3 , )andP72-D(5 , -GTACTGTAACGCAGCACAG-3 , ).

[0051] (6) RT-PCR: Amplification was performed using the Roche fluorescent quantitative PCR LightcycLer480 instrument and the optimized conditions recommended by the kit. -ΔΔCT The relative quantitative analysis of the data was performed by the method. ΔCt(1)=Ct value of the target gene in the experimental group-Ct value of the reference gene in the experimental group. ΔCt(2)=Ct value of the target gene in the control group-Ct value of the reference gene in the control group. ΔCt=ΔCt(1)-ΔCt(2).

[0052] 3. Test results and analysis The effects of drugs in experimental groups 1 to 3 on ASFV (10 3 The results of the effects of drugs in experimental groups 1 to 3 on the expression of ASFV-P72mRNA in PAM cells infected with ASFV (10 2 The results of the effect of ASFV-P72 mRNA expression in PAM cells infected with TCID50 are shown in Table 2.

[0053] Table 1 Effects of Chinese medicine on ASFV (10 3 ASFV-P72 mRNA expression in PAM cells infected with TCID50 (X±SD, n=4) Table 2 Effects of Chinese medicine on ASFV (10 2 ASFV-P72 mRNA expression in PAM cells infected with TCID50 (X±SD, n=4) It can be seen from the results in Tables 1 and 2 that the virulence of ASFV in Group 1 (Example 1), Group 2 (Example 2), and Group 3 (Example 3) is 10 3 At TCID50, the effect of reducing the expression of ASFV-p72 gene was better than that of 2 TCID50, compared with the virus control group, Examples 1 and 3 have better inhibitory effects on virus replication.

[0054] Example 5 Clinical Trial (1) Experimental groups In mid-October 2023, a pig farm in Daying Town, Weishi County, Kaifeng, had 1,705 Duchangda Sanyuan fat pigs in stock, divided into 4 buildings, with 433, 414, 427, and 431 pigs in each building, respectively, blank group, drug group (granules in Example 1), Huanglian Jiedusan group, and Wuwei Jianpi granule group. The blank group was fed with a normal basal diet, the drug group used 2 kg of feed mixed with 1 ton of feed, the Huanglian Jiedusan group used 3 kg of feed mixed with 1 ton of feed, and the Wuwei Jianpi granule group used 2 kg of feed mixed with 1 ton of feed. The specific medication conditions of each group are shown in Table 3.

[0055] Table 3 Drug use in each group of clinical trial (2) Test results and analysis When the experiment was carried out for 7 days, African swine fever infection occurred in pig farms within 5 kilometers. The pig farms then increased biosafety precautions, but abnormal pigs were detected in each group. Symptoms included: fever, weight loss, fever, multiple local skin erythema, ulcers, bulges or necrosis on the ears, abdomen, and inner thighs. Some of them developed pneumonia and arthritis, but the infection cycle was long, the mortality rate was low, and there was intermittent detoxification. The on-site veterinarian sent samples for testing and determined that it was infected with a weak strain of African swine fever. Testing and tooth extraction measures were carried out (the test found that the nucleic acid Ct value was lower than 28, which was a positive infected pig, and the pig was eliminated).

[0056] When the test was carried out to the 32nd day, there were no positive cases of infection detected for two weeks. The large group observation found that the pigs in the drug group were basically normal in feeding and spirit, with mild respiratory symptoms. Some of them coughed but no positive ASF was detected. The blank group had poor spirits, severe coughing and pneumonia symptoms, and the respiratory symptoms of the pigs survived for up to one month. The infection incidence rate was calculated by statistics = number of infected pigs / number of pigs in each group * 100%. The results showed that there were 113 pigs left in the blank group (infection rate 73.9%), 295 pigs left in the drug group (infection rate 28.7%), 254 pigs left in the Huanglian Jiedusan group (infection rate 40.5%), and 240 pigs left in the Wuwei Jianpi Granule group (infection rate 44.3%). This shows that compared with Huanglian Jiedu Powder which simply clears away heat and detoxifies and Wuwei Jianpi Granule which nourishes the spleen and stomach, the drug group can significantly reduce the clinical infection incidence of weak strains of African swine fever. This may be related to the two-pronged approach of strengthening the body and eliminating evil, invigorating Qi and consolidating the exterior, clearing the interior, and enhancing the body's oral and nasal mucosal barrier and nonspecific immune function.

[0057] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A Chinese medicine composition for preventing African swine fever attenuated virus infection, characterized in that: The weight composition of the Chinese medicine composition is: 20-40 parts of white curculigo, 5-15 parts of radix ophiopogonis, 5-15 parts of dogtail ginseng, 5-15 parts of cucurbitacin root, 5-15 parts of celery, 10-20 parts of water cypress branches, and 10-20 parts of cruciferous wormwood.

2. The Chinese medicine composition for preventing African swine fever attenuated infection according to claim 1, characterized in that: The weight composition of the traditional Chinese medicine composition is as follows: 30 parts of white curculigo, 10 parts of radix scutellariae, 10 parts of dogtail ginseng, 10 parts of cucurbitacin root, 10 parts of celery, 15 parts of water cypress branches, and 15 parts of cruciferous wormwood.

3. A drug for preventing African swine fever attenuated virus infection, characterized in that: The medicine is prepared from the extract of the traditional Chinese medicine composition according to claim 1 or 2 and pharmaceutically acceptable excipients.

4. The drug for preventing African swine fever attenuated virus infection according to claim 3, characterized in that: The dosage form of the drug is an oral dosage form.

5. The drug for preventing African swine fever attenuated virus infection according to claim 4, characterized in that: The medicine is in the form of granules, oral solution or extract.

6. The drug for preventing African swine fever attenuated virus infection according to any one of claims 3 to 5, characterized in that: The extract is a water extract of the traditional Chinese medicine composition.

7. The method for preparing the drug according to any one of claims 3 to 6, characterized in that: The steps include extracting the extract: mixing the various medicinal ingredients of the traditional Chinese medicine composition according to weight proportions, subjecting the mixture to steam explosion treatment, adding water for decoction extraction, filtering, and collecting the filtrate to obtain the extract of the traditional Chinese medicine composition.

8. The method for preparing the drug according to claim 7, characterized in that: The steam explosion treatment process has a steam pressure of 3.5 to 6 MPa and a treatment time of 4 to 8 minutes.

9. The method for preparing the drug according to claim 8, characterized in that: The filtration is carried out by using a filter press device, the pressure is 0.10-0.12 MPa, and the pore size of the filter cloth is 1000-1200 meshes.

10. The method for preparing the drug according to claim 7, characterized in that: The medicine is a granule, and the preparation steps include: concentrating the extract at 55-60° C. to prepare an extract, adding auxiliary materials, granulating, and sieving to obtain the granule.

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