Traditional Chinese medicine compound for resisting porcine epidemic diarrhea virus as well as preparation method and application of traditional Chinese medicine compound

Abstract

The invention discloses a traditional Chinese medicine compound for resisting porcine epidemic diarrhea virus as well as a preparation method and application thereof. The traditional Chinese medicine compound is prepared from the following raw material medicines in parts by weight: 100 to 150 parts of herba houttuyniae, 50 to 100 parts of herba menthae, 100 to 150 parts of flos chrysanthemi, 50 to 100 parts of folium perillae, 100 to 150 parts of fructus forsythiae and 100 to 150 parts of radix platycodonis. The traditional Chinese medicine compound has the advantages that the survival rate and the weight of the piglets infected with the PEDV are obviously improved, the body temperature rise of the piglets induced by the PEDV can be reduced, the piglet diarrhea and intestinal tissue lesion caused by the PEDV infection can be obviously relieved, and the body inflammation caused by viruses can be relieved. More importantly, the virus titer of the PEDV in intestinal tissues and excrement can be reduced, and meanwhile, the intestinal epithelial cell apoptosis induced by the PEDV can be relieved. The traditional Chinese medicine raw materials are rich in source and low in price, the production process is simple, the absorption rate of medicinal components is high, clinical use is convenient, manpower is saved, and the traditional Chinese medicine is suitable for group use.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese veterinary medicine technology, specifically to a traditional Chinese medicine compound for combating porcine epidemic diarrhea virus, its preparation method, and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is primarily caused by porcine epidemic diarrhea virus (PEDV), a member of the coronavirus family, which can infect pigs of all ages. Piglets are most susceptible, and death can occur within 2-5 days of infection, with a mortality rate reaching up to 100%. Newborn piglets under 7 days old infected with PEDV exhibit lethargy, loss of appetite or complete anorexia, vomiting, and diarrhea. Significant emaciation and dehydration occur after 3-4 days, leading to rapid death. Autopsy reveals thinning of the intestinal wall, congestion and swelling of mesenteric lymph nodes, and the presence of yellow fluid and caseous curds in the gastrointestinal tract. Histological examination shows vacuolation of jejunal epithelial cells, shortened intestinal villi, cell degeneration and necrosis, constriction of the endoplasmic reticulum, swollen and rounded mitochondria, and scattered coronavirus particles. Furthermore, only one serotype of PEDV exists, and the main sources of infection are infected and carrier pigs; fecal-oral transmission is considered the primary mode of PEDV transmission. Currently, new highly virulent strains of PEDV have evolved, and vaccinated piglets still show extremely high mortality rates, causing serious economic losses to the pig farming industry. Therefore, it is urgent to screen and develop anti-PEDV drugs.

[0003] Traditional Chinese medicine, as a natural medicine, has complex components and is rich in nutrients. It is increasingly valued for its advantages such as low toxicity and side effects, hormone-like effects, bidirectional regulatory effects, and antibacterial and antiviral properties. Rational application can have beneficial effects on the body and help improve the antiviral ability of piglets. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a traditional Chinese medicine compound for combating porcine epidemic diarrhea virus.

[0005] Another objective of this invention is to provide a method for preparing the above-mentioned traditional Chinese medicine compound for combating swine epidemic diarrhea virus.

[0006] Another object of the present invention is to provide the application of the above-mentioned traditional Chinese medicine compound for combating swine epidemic diarrhea virus.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a traditional Chinese medicine compound for combating swine epidemic diarrhea virus, the traditional Chinese medicine compound being made from the following raw materials in parts by weight: 100-150 parts of houttuynia cordata, 50-100 parts of peppermint, 100-150 parts of chrysanthemum, 50-100 parts of perilla leaf, 100-150 parts of forsythia, and 100-150 parts of platycodon grandiflorus.

[0009] Preferably, the traditional Chinese medicine compound is made from the following raw materials in parts by weight: 110-150 parts of houttuynia cordata, 50-100 parts of peppermint, 110-150 parts of chrysanthemum, 50-100 parts of perilla leaf, 110-150 parts of forsythia, and 110-150 parts of platycodon grandiflorus.

[0010] More preferably, the traditional Chinese medicine compound is made from the following raw materials in parts by weight: 110-140 parts of houttuynia cordata, 50-90 parts of peppermint, 110-140 parts of chrysanthemum, 50-90 parts of perilla leaf, 110-140 parts of forsythia, and 110-140 parts of platycodon grandiflorus.

[0011] In a further preferred embodiment, the traditional Chinese medicine compound is made from the following raw materials in parts by weight: 110-130 parts of houttuynia cordata, 50-80 parts of peppermint, 110-130 parts of chrysanthemum, 50-80 parts of perilla leaf, 110-130 parts of forsythia, and 110-130 parts of platycodon grandiflorus.

[0012] More preferably, the traditional Chinese medicine compound is made from the following raw materials in parts by weight: 120 parts of houttuynia cordata, 60 parts of peppermint, 120 parts of chrysanthemum, 60 parts of perilla leaf, 120 parts of forsythia, and 120 parts of platycodon grandiflorus.

[0013] By adopting the above technical solution, Houttuynia cordata is the principal herb in this compound formula, possessing the effects of clearing heat and detoxifying, cooling blood and promoting body fluid production. Forsythia suspensa is the assistant herb, possessing the effects of clearing heat and detoxifying, dispelling wind and expelling pathogens. The combination of the principal and assistant herbs enhances the heat-clearing and detoxifying power, while also nourishing yin and moistening dryness, cooling blood and detoxifying, and preventing heat pathogens from damaging yin and the bitter and cold nature of the herbs from damaging yin. The adjuvant herbs are peppermint, perilla leaf, and chrysanthemum, which have the effects of aromatic opening of the orifices, regulating qi and resolving dampness and turbidity. Platycodon grandiflorus is the guiding herb, serving as the envoy, promoting lung function, benefiting the throat, resolving phlegm and draining pus. The combination of these herbs clears heat and dampness, achieving the effects of clearing heat and removing dampness, cooling blood and detoxifying.

[0014] The above technical solution further reduces the weight ratio of various raw materials, saving raw material usage while ensuring efficacy.

[0015] Preferably, the dosage form of the traditional Chinese medicine compound is granules, oral liquid, etc.

[0016] In a second aspect, the present invention provides a method for preparing a traditional Chinese medicine compound for combating porcine epidemic diarrhea virus, comprising the following steps:

[0017] S1. Steam distill the houttuynia cordata and peppermint, and collect the distilled residue.

[0018] S2. Mix the dregs from step S1 with chrysanthemum, perilla leaves, forsythia, and platycodon root, add water and decoct twice. Combine the decoctions, let stand for A, filter A, concentrate to form clear extract a, add ethanol, let stand for B, filter B, collect the residue after filtration, evaporate the ethanol to remove the alcohol taste, and obtain clear extract b, which is the traditional Chinese medicine compound.

[0019] Preferably, in step S1, the houttuynia cordata and mint need to be soaked for 2 to 4 hours in advance;

[0020] Preferably, in step S1, the distillation time is 20-40 min; more preferably 30 min.

[0021] Preferably, in step S2, during the first decoction, the mass ratio of water to medicinal materials is 3 to 5:1 (preferably 5:1); during the second decoction, the mass ratio of water to medicinal materials is 2 to 4:1 (preferably 4:1).

[0022] Preferably, in step S2, the simmering time is 1.5 to 2 hours each time, and more preferably 1.5 hours;

[0023] Preferably, in step S2, the settling time A is 12 to 24 hours; more preferably, it is 12 hours.

[0024] Preferably, in step S2, the filtration A is through a 200-250 mesh sieve; more preferably, it is through a 200 mesh sieve.

[0025] Preferably, in step S2, the extract a is concentrated to a density of 1.50 to 2.0 (65°C);

[0026] Preferably, in step S2, the amount of ethanol used is such that the ethanol content reaches 70-80% (v / v), and more preferably 75% (v / v);

[0027] Preferably, in step S2, the settling time of B is 24 to 48 hours;

[0028] To better achieve the technical solution of this invention, it also includes:

[0029] S3. Mix the clear paste b from step S2 with the excipients to form a soft material, then sieve it by extrusion to form uniformly sized granules. After the wet granules are formed, dry them in time to obtain the traditional Chinese medicine compound granules.

[0030] Preferably, in step S3, the concentration of the raw material in the traditional Chinese medicine compound granules is 10-12% (w / w); more preferably 11.1% (w / w).

[0031] Preferably, in step S3, the excipients include sugar, dextrin, and wetting agent.

[0032] The sugars include sucrose, etc.

[0033] The dextrins include cyclodextrins, etc.

[0034] The wetting agent includes water, etc.

[0035] Preferably, in step S3, the mass ratio of extract b, sucrose, and cyclodextrin is 1.2–1.8:3–5:3–6; more preferably, it is 1:3:4.

[0036] Preferably, in step S3, the water content of the traditional Chinese medicine compound granules is controlled to be less than 2%.

[0037] Preferably, in step S3, the sieving is performed through a 60-80 mesh sieve; more preferably, through an 80 mesh sieve.

[0038] Preferably, in step S3, the drying temperature is 60-80°C; more preferably 60°C.

[0039] In a third aspect, this invention provides an application of a traditional Chinese medicine compound for combating porcine epidemic diarrhea virus, which is one of the following applications:

[0040] (a) Use in the preparation of drugs or feed additives for the treatment of swine epidemic diarrhea;

[0041] (b) Use in the preparation of drugs or feed additives for treating swine epidemic diarrhea virus.

[0042] Preferably, the concentration of the traditional Chinese medicine compound for combating porcine epidemic diarrhea virus in the immune-enhancing granules is 1 gram of active ingredient per 9 grams. The dosage for piglets is 1-3 g / kg body weight; more preferably, 1 g / kg body weight.

[0043] This invention prepares a traditional Chinese medicine compound using 100-150 parts by weight of Houttuynia cordata, 50-100 parts of peppermint, 100-150 parts of chrysanthemum, 50-100 parts of perilla leaf, 100-150 parts of forsythia, and 100-150 parts of platycodon. Piglets are then divided into three groups (PEDV model group, treatment group, and blank control group) according to different feeding methods. The results show that the treatment group, fed with milk powder plus the traditional Chinese medicine compound, outperformed the PEDV model group in terms of viral infection rate (assessed using PEDV-N mRNA expression level), mortality rate, diarrhea severity, and blood inflammatory markers. Therefore, this traditional Chinese medicine compound can effectively improve blood inflammatory markers and growth performance, thereby enhancing piglets' resistance to epidemic diarrhea virus infection, reducing piglet mortality, and improving economic benefits.

[0044] Furthermore, this invention also verified the antiviral effect of the traditional Chinese medicine compound in vitro (the selected research model was PEDV target cells, porcine small intestinal epithelial cells). The cells were divided into three parallel groups: a blank control group, a PEDV infection model group, and a traditional Chinese medicine compound treatment group. The results showed that, compared to the blank control group, the cells in the PEDV infection model group exhibited significant cytopathic effects accompanied by viral particle invasion. The traditional Chinese medicine compound treatment group significantly alleviated PEDV-induced cytopathic effects and reduced viral titers in the cells.

[0045] The present invention has the following advantages and effects compared with the prior art:

[0046] The traditional Chinese medicine compound of this invention is prepared by combining Houttuynia cordata, Mentha haplocalyx, Chrysanthemum morifolium, Perilla frutescens leaves, Forsythia suspensa, and Platycodon grandiflorus in specific mass proportions. This compound can effectively improve the growth performance, serum hormone levels, and blood inflammatory markers of piglets. It can also effectively reduce the viral infection rate in piglets, thereby increasing their resistance to epidemic diarrhea virus infection, thus reducing the mortality rate of infected piglets and improving economic benefits. Furthermore, the raw materials of this invention are abundant and inexpensive, and the production process of the compound is simple, resulting in a high absorption rate of the active ingredients. It is convenient for clinical use, saves manpower, and is suitable for group use. Attached Figure Description

[0047] Figure 1 This describes the effect of the traditional Chinese medicine compound in Example 3 on intestinal pathological damage in piglets.

[0048] Figure 2 This is the effect of the traditional Chinese medicine compound in Example 3 on the viability of virus-infected porcine small intestinal epithelial cells. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0050] The present invention will be further described in detail below with reference to embodiments and comparative examples.

[0051] Examples 1-4

[0052] 1. The weight proportions of each raw material in a traditional Chinese medicine compound for combating swine epidemic diarrhea virus are shown in Table 1.

[0053] Table 1 shows the weight proportions of each ingredient in a traditional Chinese medicine compound formula for combating swine epidemic diarrhea virus in Examples 1-4.

[0054] Example 1 Example 2 Example 3 Example 4 Houttuynia cordata 150 140 120 110 Mint 100 90 60 50 chrysanthemum 150 140 120 110 Perilla leaves 100 90 60 50 Forsythia 150 140 120 110 Platycodon grandiflorus 150 140 120 110

[0055] 2. A method for preparing a traditional Chinese medicine compound for combating porcine epidemic diarrhea virus.

[0056] (1) Steam distill the houttuynia cordata and mint, and collect the dregs after distillation; the houttuynia cordata and mint were soaked for 2 hours in advance, and then steam distilled for 30 minutes.

[0057] (2) Mix the dregs from step (1) with chrysanthemum, perilla leaves, forsythia, and platycodon root, add water, and decoct twice. The mass ratio of water to herbs in the first decoction is 5:1, and the mass ratio of water to herbs in the second decoction is 4:1. Each decoction lasts for 1.5 hours. Combine the two decoctions, let stand for 12 hours, and filter the supernatant through a 200-mesh sieve. The filtrate is concentrated by vacuum evaporation to a clear extract a with a density of 1.50 (65°C). Ethanol is added to make the alcohol content reach 75%, and the mixture is let stand for 48 hours and then filtered. The residue after filtration is collected, and the ethanol is evaporated to remove any alcohol odor, yielding clear extract b.

[0058] (3) Mix the clear paste b from step (2) with sucrose powder and cyclodextrin in a mass ratio of 1:3:4, add a wetting agent (water) to form a soft mass (the standard is that it can be formed into a ball by hand and crumbles when pressed). Then, extrude it through an 80-mesh sieve to make uniform granules. After the wet granules are made, dry them in time, maintaining the drying temperature at 60℃ and controlling the moisture content to within 2%. After the wet granules are dried, some may clump or stick together. Therefore, after the dry granules are cooled, they are sieved again to ensure that the granules are uniform. Finally, each 9 grams of granules contains 1 gram of raw material. The selected medicinal materials were all purchased from Kangmei Pharmaceutical Co., Ltd., sucrose powder was purchased from Hunan Xinlvfang Pharmaceutical Co., Ltd., and cyclodextrin was purchased from Shandong Zhonghuan Zhongjie Biotechnology Co., Ltd.

[0059] 3. Test materials

[0060] In vivo test of traditional Chinese medicine compound groups: The above-mentioned compound formulations with different ratios were prepared into granules (all granules in the examples were prepared using the same method). The granules were then dissolved in deionized water to make the drug concentration 1g / mL, and the treatment group was administered 1g / kg body weight.

[0061] In vitro test of the traditional Chinese medicine compound group: The above granules were dissolved in deionized water to make the drug concentration 1g / mL. The original Chinese medicine solution was obtained by autoclaving (121.3℃, 15min), centrifuged at 4℃ (8000rpm), and the supernatant was collected. The solution was filtered through a 0.22μm filter for sterilization, aliquoted, and stored at -80℃ for later use.

[0062] 4. Test methods

[0063] 4.1 In vivo group

[0064] Three-day-old piglets from pig farms that had never been vaccinated with porcine epidemic diarrhea virus (PEDV) were selected for in vivo animal experiments using a traditional Chinese medicine compound. Each experimental group of piglets was randomly divided into three groups of 30 piglets each: a blank control group, a PEDV model group, and a traditional Chinese medicine compound treatment group (Table 2). After one day of acclimatization at the experimental site, piglets in the PEDV model group and the traditional Chinese medicine compound treatment group were orally challenged with 5 mL of PEDV virus solution (10 mL / min). 5.48 TCID 50 / mL)(Virus fluid mixed with 180mL of milk powder for challenge). The traditional Chinese medicine compound treatment group received oral medication twice daily, morning and evening, one day after challenge. The dosage was calculated based on 1g / kg / body weight, converted to the required granule dosage per piglet, and then dissolved in 180mL of milk powder before feeding. Piglets in the blank control group and PEDV model group received oral 180mL of milk powder solution (20g milk powder dissolved thoroughly in 180mL of water). The experiment lasted 5 days. Note: Piglet milk powder was purchased from Sino-American Puke Biotechnology Co., Ltd. (Zhengzhou, Henan).

[0065] 4.2 In vitro group

[0066] Porcine small intestinal epithelial cells IPEC-J2 (purchased from Beijing Baipusaisi Biotechnology Co., Ltd.) were used to set up three parallel experimental groups: a blank control group, a PEDV infection model group (PEDV (0.1 MOI), and a traditional Chinese medicine compound treatment group (1 g / mL traditional Chinese medicine compound) (Table 3). Two hours after PEDV infection (at which point the cell density was approximately 50%), the virus solution in the PEDV infection model group and the traditional Chinese medicine compound treatment group was discarded, and the cells were washed twice with PBS. Fresh culture medium was then added. The traditional Chinese medicine compound treatment group received a 1 g / mL traditional Chinese medicine compound granule solution as treatment. Cells were collected after 24 hours. The PEDV-N gene (with β-actin as the internal reference gene) was detected by RT-PCR, and cell viability after drug treatment was detected by CCK8 assay.

[0067] Table 2 Grouping of In Vitro Tests on Piglets

[0068] Group Feeding method Number of piglets (units) control group milk powder 30 PEDV model group Milk powder + PEDV virus solution 30 Treatment group Milk powder + PEDV virus solution + 1g / kg traditional Chinese medicine compound granule solution 30

[0069] Table 3 Grouping of porcine small intestinal epithelial cells in vitro experiments

[0070] Group Training methods control group culture medium PEDV model group Culture medium + PEDV virus solution Treatment group Culture medium + PEDV virus solution + 1g / mL traditional Chinese medicine compound granule solution

[0071] The test indicators were determined as follows:

[0072] (1) Mortality rate

[0073] The mortality rate and weight of piglets in each group are recorded at 15:00 every day. Mortality rate (%) = Number of dead piglets / Initial number of piglets in each group.

[0074] (2) Clinical symptom observation and scoring, stool viscosity score, and weight and body temperature monitoring.

[0075] Clinical symptoms of piglets in each group were collected and scored according to standards at 3:00 PM daily. Fecal consistency of piglets in each group was also scored at 3:00 PM daily. Weight refers to the piglet's weight in kilograms. Temperature refers to the rectal temperature of the piglets.

[0076] (3) Detection of intestinal pathological damage

[0077] On the last day of the trial, all piglets were euthanized and their jejunum and ileum were collected for sectioning and pathological observation.

[0078] (4) Effects of traditional Chinese medicine compound on PEDV virus RNA expression in piglets' intestines

[0079] At the end of the trial period, three piglets were randomly selected from each group, and their intestines and fresh feces were collected. RNA was routinely extracted from the intestinal tissue and feces, and relative quantitative detection of viral RNA was performed.

[0080] (5) Effects of traditional Chinese medicine compound on blood inflammatory markers in PEDV-infected piglets

[0081] On the last day of the trial, blood samples were collected from all groups of piglets for analysis of inflammatory markers, including IL-6, IL-8, and TNF-α.

[0082] (6) Detection of viral titer in porcine small intestinal epithelial cells

[0083] Porcine small intestinal epithelial cells were treated according to the grouping and administration methods specified in the experimental protocol, and viral titers were detected by plaque formation (PFU method).

[0084] (7) Detection of apoptosis rate of porcine small intestinal epithelial cells

[0085] Porcine small intestinal epithelial cells were treated according to the grouping and administration methods specified in the experimental protocol, and the apoptosis rate was detected by flow cytometry.

[0086] Test judgment criteria:

[0087] Criteria for determining cure: significant improvement in mortality, disappearance of clinical symptoms, reduction in enterovirus load, and significant reduction in intestinal damage;

[0088] Effective criteria: Some clinical symptoms have improved, appetite has returned, and enterovirus levels have decreased, but not significantly;

[0089] Invalid criteria: death or failure to improve clinical symptoms.

[0090] 5. Test Results

[0091] The statistical software SPSS 22.0 was used to perform one-way ANOVA and Duncan's method to analyze the significance of differences among multiple groups of experimental data. All data are expressed as mean ± standard deviation. The results are shown in Tables 4 to 9.

[0092] 5.1 Effect of a traditional Chinese medicine compound for combating porcine epidemic diarrhea virus (PEDV) in Examples 1-4 on the efficacy of PEDV-infected pigs.

[0093] Table 4. Performance analysis of traditional Chinese medicine compound formulas against porcine epidemic diarrhea virus in Examples 1-4 (only cases in the treatment group are counted).

[0094]

[0095] As can be seen from Examples 1-4 and Table 4, the cure rate and total effective rate of the traditional Chinese medicine compound against porcine epidemic diarrhea virus (PEDV) in experimental groups 1-5 were higher than those in the blank control group (group 5). This indicates that the traditional Chinese medicine compound against PEDV in this invention has the effects of clearing heat and detoxifying, and resisting PEDV virus. Furthermore, the antiviral effect of the traditional Chinese medicine compound in Example 3 is superior to that in other groups. Specific antiviral data for Example 3 are attached below.

[0096] 5.2 Effect of Example 3 on Mortality Rate of PEDV-Infected Pigs

[0097] Table 5. Effects of Traditional Chinese Medicine Compound on Mortality in PEDV-Infected Pigs

[0098] Control group (%) PEDV model group (%) Treatment group (%) Day 1 0 0 0 Day 2 0 20 20 Day 3 0 30 30 Day 4 0 70 30 Day 5 0 100 30

[0099] As shown in Table 5, the mortality rate of piglets in the three groups was the same on the first day of the experiment. Compared with the control group, there was no significant difference in mortality rate between the PEDV model group and the treatment group on days 2 and 3 of the experiment. On days 4 and 5, the mortality rate of piglets in the PEDV model group was significantly higher than that in the control group and the treatment group.

[0100] 5.3 Effects of Example 3 on clinical symptom scores, fecal viscosity scores, body weight, and body temperature in piglets

[0101] Table 6. Effects of Traditional Chinese Medicine Compound on Clinical Symptom Scores, Fecal Viscosity Scores, Body Weight, and Body Temperature in Piglets.

[0102]

[0103] Note: Different letters in each column indicate significant differences when comparing the same date (P<0.05).

[0104] Table 6 shows that, compared with the control group, the PEDV model group and the treatment group had significant differences in clinical symptom scores, fecal viscosity scores, body weight, and body temperature (P<0.05). However, the treatment group showed a significant decrease in piglet temperature (P<0.05) and a significant increase in piglet body weight (P<0.05), as well as a significant reduction in clinical symptom scores and fecal viscosity scores compared with the PEDV model group. This demonstrates that the traditional Chinese medicine compound alleviated the clinical symptoms caused by PEDV infection to a certain extent.

[0105] 5.4 Example 3: Detection of intestinal pathological damage in piglets

[0106] Depend on Figure 1 It was found that the lesions were mainly concentrated in the jejunum and ileum of the pigs treated with the virus. Hematoxylin and eosin (HE) staining results showed that, compared with the blank control group, the jejunum and ileum villi of the piglets in the PEDV model group were ruptured, and vacuolar degeneration was observed in the intestinal mucosal epithelial cells. Although inflammatory infiltration was present in the intestinal pathological sections of the traditional Chinese medicine compound treatment group, the lesions were significantly milder than those in the model group.

[0107] 5.5 Effect of Example 3 on the viability of porcine small intestinal epithelial cells

[0108] Depend on Figure 2 It was found that, compared with the blank control group, the cell viability of the PEDV model group was significantly reduced (P<0.0001), while the cell viability of the treatment group was significantly restored compared with the PEDV model group (P<0.0001). This indicates that the traditional Chinese medicine compound significantly alleviated PEDV-induced cell death.

[0109] 5.6 Effect of Example 3 on the expression of PEDV virus N protein mRNA in the intestines and feces of piglets

[0110] Table 7. Relative expression levels of PEDV virus N protein mRNA in piglet intestines and feces.

[0111] Group / Part jejunum ileum stool control group <![CDATA[1.0±0.02 c ]]> <![CDATA[1.0±0.15 c ]]> <![CDATA[1.0±0.14 c ]]> PEDV model group <![CDATA[6.7±1.13 a ]]> <![CDATA[5.0±0.03 a ]]> <![CDATA[5.8±0.31 a ]]> Treatment group <![CDATA[6.0±1.74 b ]]> <![CDATA[2.5±0.14 b ]]> <![CDATA[3.0±0.33 b ]]>

[0112] Note: Different letters in each column indicate significant differences (P<0.05).

[0113] Table 7 shows that, compared with the control group, the mRNA expression level of PEDV N protein in the PEDV model group was significantly increased. In contrast to the PEDV model group, the treatment group showed a significant decrease in the mRNA expression level of PEDV N protein at different locations, indicating that the traditional Chinese medicine compound inhibited PEDV virus replication and reduced the virus infection rate in piglets.

[0114] 5.7 Effects of Example 3 on inflammatory markers in porcine blood

[0115] Table 8. Concentration of inflammatory factors in porcine blood

[0116] Groups / Indicators IL-6 (pg / mL) IL-8 (pg / mL) TNF-α (pg / mL) control group <![CDATA[7.52±0.17 c ]]> <![CDATA[50.17±1.74 c ]]> <![CDATA[17.58±0.16 c ]]> PEDV model group <![CDATA[157±2.58 a ]]> <![CDATA[134±1.08 a ]]> <![CDATA[35.32±0.12 a ]]> Treatment group <![CDATA[60.41±1.86 b ]]> <![CDATA[67.51±0.26 b ]]> <![CDATA[18.16±0.02 b ]]>

[0117] As shown in Table 8, compared with the control group, the level of blood inflammatory factors in the PEDV model group was significantly increased (P<0.05), and compared with the PEDV model group, the level of blood inflammatory factors in the treatment group was significantly decreased (P<0.05), indicating that the PEDV virus-induced inflammatory state was significantly improved under the treatment of traditional Chinese medicine compound.

[0118] 5.8 Effect of Example 3 on Virus Titer in Porcine Small Intestinal Epithelial Cells

[0119] Table 9 Virus titers in porcine small intestinal epithelial cells

[0120] Group Jejunum / Lg(pfu / mL) Ileum / Lg(pfu / mL) control group <![CDATA[0 c ]]> <![CDATA[0 c ]]> PEDV model group <![CDATA[4.5±0.32 a ]]> <![CDATA[4.7±0.08 a ]]> Treatment group <![CDATA[3.2±0.17 b ]]> <![CDATA[2.5±0.13 b ]]>

[0121] Note: Different letters in each column indicate significant differences (P<0.05).

[0122] As shown in Table 9, compared with the control group, the viral titer in the PEDV model group was significantly increased (P<0.05), and compared with the PEDV model group, the viral titer in the treatment group was significantly decreased (P<0.05), indicating that viral replication was significantly inhibited under the treatment of traditional Chinese medicine compound.

[0123] 5.9 Effect of Example 3 on Apoptosis Rate of Porcine Small Intestinal Epithelial Cells

[0124] Table 10 Apoptosis rate of porcine small intestinal epithelial cells

[0125] Group jejunum(%) Ileum (%) control group <![CDATA[0.47±0.03 b ]]> <![CDATA[0.33±0.02 b ]]> PEDV model group <![CDATA[0.82±0.12 a ]]> <![CDATA[0.79±0.14 a ]]> Treatment group <![CDATA[0.15±0.07 c ]]> <![CDATA[0.26±0.04 c ]]>

[0126] Note: Different letters in each column indicate significant differences (P<0.05).

[0127] As shown in Table 10, compared with the control group, the apoptosis rate in the PEDV model group was significantly increased (P<0.05), while the apoptosis rate in the treatment group was significantly reduced compared with the PEDV model group. This indicates that the traditional Chinese medicine compound significantly alleviated PEDV-induced apoptosis.

[0128] From Tables 1-10 and Figures 1-2 It is evident that the traditional Chinese medicine compound of the present invention for improving piglet resistance to PEDV infection significantly improves the survival rate and weight of PEDV-infected piglets, reduces PEDV-induced fever in piglets, and significantly improves virus-induced inflammation. Furthermore, this traditional Chinese medicine compound can significantly alleviate diarrhea and intestinal tissue lesions in piglets caused by PEDV infection. More importantly, this traditional Chinese medicine compound can reduce the viral titer of PEDV in intestinal tissue and feces, and simultaneously alleviate PEDV-induced intestinal epithelial cell apoptosis. Moreover, this traditional Chinese medicine compound is safe and effective in use.

[0129] In summary, compared with the PEDV model group, the treatment group fed with this traditional Chinese medicine compound showed significant effects in improving the survival rate of infected piglets, reducing piglet temperature, alleviating clinical symptoms, reducing PEDV titers, and improving virus-induced inflammation. Furthermore, this traditional Chinese medicine compound does not contain antibiotics, will not lead to drug resistance, and will not pose a threat to food safety. Therefore, this traditional Chinese medicine compound can enhance piglets' resistance to PEDV infection and treat infection-induced clinical symptoms, providing a new approach for antibiotic-free, green, and healthy farming models.

[0130] Furthermore, through multiple experiments, the inventors discovered that the traditional Chinese medicine compound group, weighed in parts as follows: 100-150 parts of Houttuynia cordata, 50-100 parts of Mentha haplocalyx, 100-150 parts of Chrysanthemum morifolium, 50-100 parts of Perilla frutescens, 100-150 parts of Forsythia suspensa, and 100-150 parts of Platycodon grandiflorus. With the total amount of the compound being the same, minor variations in the amount of each herb had minimal impact on the experimental results. Therefore, the embodiments of this invention detail the experimental results using 120g of Houttuynia cordata, 60g of Mentha haplocalyx, 120g of Chrysanthemum morifolium, 60g of Perilla frutescens, 120g of Forsythia suspensa, and 120g of Platycodon grandiflorus. After dosage screening, it was determined that administering the traditional Chinese medicine compound to piglets at a dose of 1g / kg via gavage maximizes the cost-effectiveness of the medicine.

[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine compound against porcine epidemic diarrhea virus, characterized in that: The traditional Chinese medicine compound is made of raw medicinal materials in the following weight parts: 100-150 parts of Houttuynia cordata, 50-100 parts of Mentha haplocalyx, 100-150 parts of Chrysanthemum morifolium, 50-100 parts of Perilla frutescens, 100-150 parts of Forsythia suspensa, and 100-150 parts of Platycodon grandiflorum; ​ Further, the traditional Chinese medicine compound is made of raw medicinal materials in the following weight parts: 110-150 parts of Houttuynia cordata, 50-100 parts of Mentha haplocalyx, 110-150 parts of Chrysanthemum morifolium, 50-100 parts of Perilla frutescens, 110-150 parts of Forsythia suspensa, and 110-150 parts of Platycodon grandiflorum; Further, the traditional Chinese medicine compound is made of raw medicinal materials in the following weight parts: 110-140 parts of Houttuynia cordata, 50-90 parts of Mentha haplocalyx, 110-140 parts of Chrysanthemum morifolium, 50-90 parts of Perilla frutescens, 110-140 parts of Forsythia suspensa, and 110-140 parts of Platycodon grandiflorum; Further, the traditional Chinese medicine compound is made of raw medicinal materials in the following weight parts: 110-140 parts of Houttuynia cordata, 50-90 parts of Mentha haplocalyx, 110-140 parts of Chrysanthemum morifolium, 50-90 parts of Perilla frutescens, 110-140 parts of Forsythia suspensa, and 110-140 parts of Platycodon grandiflorum; Further, the traditional Chinese medicine compound is made of raw medicinal materials in the following weight parts: 110-140 parts of Houttuynia cordata, 50-90 parts of Mentha haplocalyx, 110-140 parts of Chrysanthemum morifolium, 50-90 parts of Perilla frutescens, 110-140 parts of Forsythia suspensa, and 110-140 parts of Platycodon grandiflorum; 2. The traditional Chinese medicine compound against porcine epidemic diarrhea virus according to claim 1, characterized in that: The dosage form of the traditional Chinese medicine compound is granules or oral liquid.

3. The method for preparing the traditional Chinese medicine compound for resisting porcine epidemic diarrhea virus according to any one of claims 1-2, characterized in that: The method comprises the following steps: S1, steam distillation of Houttuynia cordata and Mentha haplocalyx, and collecting the residue after distillation; S2, mixing the residue in step S1 with Chrysanthemum morifolium, Perilla frutescens, Forsythia suspensa, and Platycodon grandiflorum with water, and decocting twice, combining the decocting liquid, standing A, filtering A, concentrating to form clear paste a, adding ethanol, standing B, filtering B, collecting the remaining filtered material, and volatilizing ethanol to remove the alcohol taste to obtain clear paste b, which is the traditional Chinese medicine compound.

4. The preparation method according to claim 3, characterized in that: In step S1, Houttuynia cordata and Mentha haplocalyx need to be soaked for 2-4 hours in advance; In step S1, the distillation time is 20-40 min.

5. The preparation method according to claim 3, characterized in that: In step S2, the mass ratio of water to medicinal materials is 3-5:1 in the first decoction, and 2-4:1 in the second decoction; In step S2, the decoction time is 1.5-2 hours each time; In step S2, the amount of ethanol is used to make the ethanol content reach 70-80% v / v.

6. The preparation method according to claim 3, characterized in that: In step S2, the standing time of A is 12-24 hours; In step S2, the filtering of A is through a 200-250 mesh sieve; In step S2, the standing time of B is 24-48 hours.

7. The method of any one of claims 3 to 6, wherein the method further comprises, Further comprising: S3, mixing clear paste b in step S2 with excipients, making soft material, and then sieving in an extrusion manner to make uniform granules, and drying the wet granules in time to obtain traditional Chinese medicine compound granules.

8. The preparation method according to claim 7, characterized in that: The concentration of the raw medicine in the traditional Chinese medicine compound granules is 10-12% w / w in step S3; The auxiliary materials include sugar, dextrin and wetting agent in step S3; The water content in the traditional Chinese medicine compound granules is controlled within 2% in step S3; The sieving is 60-80 mesh sieving in step S3; The drying temperature is 60-80℃ in step S3.

9. The application of the traditional Chinese medicine compound for resisting porcine epidemic diarrhea virus according to claim 1 or 2, characterized in that, For one of the following applications: (a) the application in preparing a medicine or feed additive for treating porcine epidemic diarrhea; (b) the application in preparing a medicine or feed additive for resisting porcine epidemic diarrhea virus.

10. Use according to claim 9, characterized in that, The administration amount of the traditional Chinese medicine compound for resisting porcine epidemic diarrhea virus is 1-3g per kilogram of piglets.

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