A pharmaceutical composition for treating piglet diarrhea and a preparation method thereof

Abstract

The present application belongs to the technical field of medicine, and particularly relates to a kind of medicine composition for treating pig diarrhea and a preparation method thereof.The medicine composition for treating pig diarrhea of the present application comprises the following raw materials: echinocystic acid, and veratrum album cyclic peptide B, and the mass ratio of the echinocystic acid and the veratrum album cyclic peptide B is 1:0.15-0.25.The present application protects a kind of medicine composition for treating pig diarrhea, and experiments prove that the composition can inhibit the proliferation of porcine epidemic diarrhea virus, the echinocystic acid has good inhibitory effect on the porcine epidemic diarrhea virus, the addition of the veratrum album cyclic peptide B further improves the inhibitory effect, and has obvious synergistic effect.The composition has good application prospect in the treatment of diarrhea caused by porcine epidemic diarrhea virus, is high in safety, free of toxic side effects, and is conducive to improving the economic benefits of pig breeding.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for treating swine diarrhea and its preparation method. Background Technology

[0002] Swine diarrhea is a common digestive tract disease in pig farming, with porcine epidemic diarrhea virus (PEDV) causing the most serious harm. This disease is characterized by rapid transmission, high morbidity, and high mortality. It primarily infects the epithelial cells of the small intestinal villi, leading to malabsorption and clinical manifestations such as acute diarrhea, vomiting, and dehydration. Mortality rates are extremely high in piglets under 7 days old. While mortality rates are lower in adult pigs, they often experience loss of appetite and decreased production performance, causing significant economic losses to the global pig farming industry. Furthermore, PEDV infection weakens the pig's immune system, making them susceptible to secondary bacterial infections such as E. coli and Salmonella, leading to mixed infections, worsening the condition, and increasing the difficulty of treatment.

[0003] Currently, the prevention and control of porcine epidemic diarrhea (PED) mainly relies on vaccination and biosecurity measures. However, due to the high mutation rate of PEDV and the diversity of circulating strains, existing vaccines are mainly developed for specific circulating strains, resulting in deficiencies such as delayed immune protection and limited protective spectrum, making it difficult to achieve comprehensive and effective prevention and control. Clinical treatment lacks highly effective and specific antiviral drugs, and is mostly based on symptomatic and supportive treatment, such as electrolyte supplementation, antidiarrheal medication, and antibacterial and anti-inflammatory drugs. These measures only alleviate surface symptoms such as diarrhea and dehydration, and cannot fundamentally inhibit viral proliferation, leading to frequent relapses.

[0004] While some research reports exist on anti-PEDV drugs in existing technologies, single-component drugs often suffer from insufficient inhibitory efficiency and limited target specificity. Traditional Chinese medicine and its extracts have attracted widespread attention in the field of veterinary antiviral drug development due to their abundant resources, relatively low cost, minimal toxicity and side effects, and low likelihood of inducing drug resistance.

[0005] Hypochondrial acid is a natural triterpenoid compound isolated from certain medicinal plants. Modern pharmacological studies have shown that hypochondrial acid possesses various biological activities, including anti-inflammatory, antioxidant, antitumor, antiviral, and immunomodulatory effects. Vaccaria segetalis cyclic peptide B is a cyclic peptide compound isolated from the traditional Chinese medicine Vaccaria segetalis. Existing studies have shown that it has good immunomodulatory activity, promoting the proliferation of immune cells and enhancing immunity. However, its application in combination with hypochondrial acid in combating porcine epidemic diarrhea virus (PEDV) remains poorly investigated. Summary of the Invention

[0006] The primary objective of this invention is to provide a pharmaceutical composition for treating swine diarrhea. This composition uses spiculic acid and Wangbuliuxing cyclic peptide B as its core active ingredients. By optimizing the mass ratio of the two, a synergistic effect is achieved, which can significantly inhibit the proliferation of swine epidemic diarrhea virus. The therapeutic effect is superior to that of a single ingredient, and it is safe and has no toxic side effects. It can effectively treat swine epidemic diarrhea and provides an efficient and safe veterinary drug option for the clinical treatment of swine epidemic diarrhea.

[0007] The second objective of this invention is to provide a method for preparing a pharmaceutical composition for treating swine diarrhea. This method is simple, easy to operate, and suitable for large-scale production, thus facilitating the promotion and application of the pharmaceutical composition.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A pharmaceutical composition for treating swine diarrhea comprises the following raw materials: spiculin and Wangbuliuxing cyclic peptide B, wherein the mass ratio of spiculin to Wangbuliuxing cyclic peptide B is 1:(0.15-0.25).

[0009] Furthermore, the mass ratio of the strychnine and Wangbuliuxing cyclic peptide B is 1:0.2.

[0010] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the dosage form of the pharmaceutical composition is any one of tablets, powders, granules, oral liquids, and injections.

[0012] A method for preparing a pharmaceutical composition for treating swine diarrhea includes the following steps: According to the formula, mix the cystic acid and Wangbuliuxing cyclic peptide evenly, and add pharmaceutically acceptable excipients to obtain the final product.

[0013] Furthermore, the pharmaceutically acceptable excipients include at least one of lactose, soluble starch, and wheat bran.

[0014] Compared with the prior art, the main advantages of the present invention are as follows: 1. The pharmaceutical composition for treating swine diarrhea provided by this invention has been proven through testing to have the following technical effects: (1) Significant synergistic antiviral effect: This composition can inhibit the proliferation of porcine epidemic diarrhea virus. Spicoic acid has a good inhibitory effect on porcine epidemic diarrhea virus. The addition of Wangbuliuxing cyclic peptide B can further enhance its inhibitory effect. The two form a significant synergistic effect. Compared with single-component drugs, the inhibitory effect on PEDV is significantly improved.

[0015] (2) The composition has good application prospects in the treatment of diarrhea caused by porcine epidemic diarrhea virus. It is safe, has no toxic side effects, and is conducive to improving the economic benefits of pig farming.

[0016] 2. The preparation process of the pharmaceutical composition for treating swine diarrhea provided by the present invention is simple, adopts conventional formulation process, the raw materials are readily available, the operation is convenient, the cost is controllable, and it is suitable for large-scale production.

[0017] 3. The pharmaceutical composition for treating swine diarrhea provided by this invention can be made into various dosage forms such as tablets, powders, and granules, and the administration method can be flexibly selected according to the age, weight and farming environment of the pigs. Attached Figure Description

[0018] Figure 1 These are Western blot detection results of Examples 1-3 and Comparative Examples 1-2 of the present invention; Figure 2 Clinical scores of piglets in Examples 1-3 and Comparative Examples 1-2 of this invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0020] Example 1 A pharmaceutical composition for treating swine diarrhea comprises the following raw materials: spiculic acid and Wangbuliuxing cyclic peptide B in a mass ratio of 1:0.2.

[0021] A method for preparing a pharmaceutical composition for treating swine diarrhea includes the following steps: According to the formula, spiculin and Wangbuliuxing cyclic peptide are mixed evenly, and then 25% lactose and 25% soluble starch of the total mass of spiculin and Wangbuliuxing cyclic peptide are added. The mixture is then granulated, dried, and the granule dosage form is obtained.

[0022] Example 2 A pharmaceutical composition for treating swine diarrhea comprises the following raw materials: spiculic acid and Wangbuliuxing cyclic peptide B in a mass ratio of 1:0.15.

[0023] A method for preparing a pharmaceutical composition for treating swine diarrhea includes the following steps: According to the formula, spiculin and Wangbuliuxing cyclic peptide are mixed evenly, and then 20% of the total mass of lactose and 30% of soluble starch are added. The mixture is mixed evenly, compressed into tablets, and dried to obtain the tablet dosage form.

[0024] Example 3 A pharmaceutical composition for treating swine diarrhea comprises the following raw materials: spiculic acid and Wangbuliuxing cyclic peptide B in a mass ratio of 1:0.25.

[0025] A method for preparing a pharmaceutical composition for treating swine diarrhea includes the following steps: According to the formula, mix the cystic acid and Wangbuliuxing cyclic peptide evenly, add 50% of the total mass of cystic acid and Wangbuliuxing cyclic peptide with wheat bran, mix evenly, and the powder dosage form is obtained.

[0026] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Wangbuliuxing cyclic peptide B is not added to the pharmaceutical composition; otherwise, they are consistent with Example 1.

[0027] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that no styracimenic acid is added to the pharmaceutical composition; otherwise, they are consistent with Example 1.

[0028] Test case To verify the safety and therapeutic efficacy of the pharmaceutical composition of the present invention, the following tests were conducted: Example 1: CCK8 assay for the toxicity of the drug composition to Vero cells (African green monkey kidney cells). The spiculin and Wangbuliuxing cyclic peptide B of this invention were mixed at a mass ratio of 1:0.2, and a stock solution of 2 mg / mL was prepared using medical-grade dimethyl sulfoxide (DMSO) for later use. Vero cells in the logarithmic growth phase were then cultured at a concentration of 1 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. Once the cells had adhered and grown into a monolayer, the original cell culture medium was removed and replaced with DMEM medium containing different concentrations of the drug (spiculin and Wangbuliuxing cyclic peptide B in a mass ratio of 1:0.2). The final drug concentrations in each well were controlled at 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL, and 80 µg / mL. A cell control group (cells and an equal volume of DMEM medium) and a blank control group (only an equal volume of DMEM medium) were set up. The cells were then cultured at 37°C in a 5% CO2 incubator for another 48 h. After the culture period, CCK-8 solution was added to each well, and the cells were cultured for 1 h. The absorbance of each well was measured at 450 nm to calculate the cell viability.

[0029] Cell viability (%) = (OD450 value of experimental group - OD450 value of blank control group) / (OD450 value of cell control group - OD450 value of blank control group) × 100%. The experimental results are shown in Table 1.

[0030] Table 1 As shown in Table 1, the combined use of styracidium and Vaccaria segetalis cyclic peptide B of the present invention resulted in a Vero cell survival rate of over 99% within a concentration range of 5 μg / mL to 80 μg / mL, with no significant difference compared to the cell control group. This indicates that the pharmaceutical composition of the present invention has no cytotoxicity to Vero cells and exhibits good safety, providing a safety guarantee for its subsequent application.

[0031] Experimental Example 2: Validation of the antiviral effect of the drug composition 1. Experimental materials: Vero cells, PEDV strain, spiculic acid, Wangbuliuxing cyclic peptide B, etc.; 2. Experimental Groups: Virus control group: Vero cells + PEDV strain; Example 1 group: Vero cells + PEDV strain + spiculic acid and Wangbuliuxing cyclic peptide B were mixed at a mass ratio of 1:0.2, with a final concentration of 20 mg / mL; Example 2 group: Vero cells + PEDV strain + styracidium and Vaccaria segetalis cyclic peptide B mixed at a mass ratio of 1:0.15, with a final concentration of 20 mg / mL; Example 3 group: Vero cells + PEDV strain + styracidium and Vaccaria segetalis cyclic peptide B mixed at a mass ratio of 1:0.25, with a final concentration of 20 mg / mL; Comparative Example 1: Vero cells + PEDV strain + spiculic acid (final concentration 20 mg / mL). Comparative Example 2: Vero cells + PEDV strain + Vaccaria segetalis cyclic peptide B (final concentration 20 mg / mL). 3. Test methods: Vero cells were divided into 2×10 5 Cells / mL were seeded into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until a monolayer was formed. PEDV strain (multiple of infection of 0.1 MOI) was added and incubated at 37°C for 2 hours. After removing the supernatant, uninvaded virus particles were washed with PBS, and then the corresponding drugs were added according to Examples 1-3 and Comparative Examples 1-2, and cultured for another 16 hours. Viral supernatant from each group was collected and the viral titer (TCID50) was tested.

[0032] Uninfected Vero cells were divided into groups of 2 × 10⁻⁶. 5Virus supernatant was inoculated at a rate of 1 / mL into 96-well plates. After a confluent monolayer was formed, supernatant from Examples 1-3 and Comparative Examples 1-2 was inoculated at 10-fold serial dilutions 16 hours after infection. Eight replicates were set up for each dilution. Supernatant from PEDV-infected cells without drug treatment was used as a viral control group. The inoculated 96-well plates were incubated at 37°C in a 5% CO2 incubator. Cytopathic effect (CPE) was observed daily, and the number of wells with CPE was recorded until the number of wells with CPE ceased to increase. The number of wells with CPE was recorded, and the TCID50 of the virus was calculated. The results are shown in Table 2.

[0033] Table 2 As shown in Table 2, the virus titer was the highest in the virus control group, indicating that PEDV proliferated normally in Vero cells. The virus titers in the Example 1-3 groups were significantly lower than those in the Comparative Example 1-2 groups, with the lowest virus titer in the Example 1 group. This indicates that the drug composition of the present invention (spicate acid + Wangbuliuxing cyclic peptide B) has a significantly better inhibitory effect on PEDV than the single component, and the two have a significant synergistic effect, which can effectively inhibit the proliferation of PEDV.

[0034] Experimental Example 3: Western blot detection of PEDV-N protein levels Vero cells were 1×10 5Cells were seeded at a density of 1 / mL in 96-well cell culture plates and cultured to a monolayer in a 37°C, 5% CO2 incubator. PEDV strain (multiple of infection of 0.1 MOI) was added and incubated at 37°C for 2 hours. After removing the supernatant, uninvaded virus particles were washed with PBS. Culture medium containing the corresponding drug for each group was added to each well, forming the experimental groups. Specifically, Example 1 group: spiculin and Vaccaria segetalis cyclic peptide B were mixed at a mass ratio of 1:0.2 to a final concentration of 20 mg / mL; Example 2 group: spiculin and Vaccaria segetalis cyclic peptide B were mixed at a mass ratio of 1:0.15 to a final concentration of 20 mg / mL; Example 3 group: spiculin and Vaccaria segetalis cyclic peptide B were mixed at a mass ratio of 1:0.25 to a final concentration of 20 mg / mL; Comparative Example 1: spiculin (final concentration 20 mg / mL); Comparative Example 2: Vaccaria segetalis cyclic peptide B (final concentration 20 mg / mL). A blank control group was set up, which did not receive drug treatment after PEDV infection. After 16 h of culture, the culture medium was discarded, cells were collected, washed twice with PBS, and then lysed with cell lysis buffer. Cells were centrifuged at 12000 rpm for 15 min at 4 °C to obtain the supernatant protein. The protein concentration of each group was determined using the BCA method. The loading volume of each group was adjusted to be consistent. After protein denaturation, the cells were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h. PEDV-N primary antibody (1:1000 dilution) and β-actin primary antibody (1:5000 dilution) were added, and the membranes were incubated overnight at 4 °C. The cells were washed three times with TBST buffer for 10 min each time. Secondary antibody (1:2000 dilution) was added, and the membranes were incubated at room temperature for 1 h. The cells were washed three times with TBST buffer for 10 min each time. After development, the expression level of PEDV-N protein was analyzed, with β-actin used as an internal control protein. The experimental results are shown below. Figure 1 .

[0035] PEDV-N protein is a core structural protein of porcine epidemic diarrhea virus (PEDV), and its expression level directly reflects the degree of viral infection and proliferation; the lower the expression level, the more significant the inhibition of viral proliferation. Figure 1 It can be seen that the PEDV-N protein expression level was the highest in the virus control group. Compared with the virus control group, the drug compositions obtained in Examples 1-3 of this invention can downregulate the protein expression level of PEDV-N, indicating that the drug compositions of this invention can inhibit viral infection. Among them, compared with Example 1, the PEDV-N protein expression levels in Comparative Examples 1 and 2 were higher, further confirming that the synergistic effect of schizocarpine and Wangbuliuxing cyclic peptide B in the drug compositions of this invention can significantly enhance the antiviral effect and effectively inhibit PEDV infection and proliferation.

[0036] Example 4: In vivo test to verify the therapeutic effect of traditional Chinese medicine composition on swine epidemic diarrhea. Healthy 7-day-old piglets were selected as experimental animals and randomly divided into four groups: Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Blank Control Group, and Infection Group, with 6 piglets in each group. Except for the Blank Control Group, the piglets were divided into four groups according to the following ratio: 10... 5 TCID 50 The experimental piglets were given a single oral dose of PEDV virus at a dose of 1.0 mL. 12 hours after challenge, the piglets in Examples 1-3 and Comparative Examples 1-2 were given the corresponding drug composition by gavage at a dose of 10 mg / kg. The blank control group was given physiological saline by gavage. The treatment was carried out every 6 hours for 66 hours.

[0037] After treatment, the clinical symptoms of piglets in each group were systematically observed and quantitatively scored. The scoring system included four core indicators: mental state, feeding ability, severity of diarrhea, and coat condition. Each indicator was scored from 1 to 4 points. Specific scoring criteria were as follows: 1 point indicated no abnormality and normal physiological state; 2 points indicated mild symptoms that did not affect normal physiological activities; 3 points indicated severe symptoms that significantly impacted the piglet's physiological state; if a piglet died, all clinical indicator scores were uniformly recorded as 4 points. The total clinical score for a single piglet was the sum of the scores from the four indicators. Results are shown below. Figure 2 .

[0038] Depend on Figure 2 It can be seen that the clinical score of the infection group was the highest, indicating that the challenge was successful. The clinical scores of the Example 1-3 groups were close to those of the blank control group and significantly lower than those of the Comparative Example 1-2 groups. Among them, the diarrhea rate of Example 1 group was the lowest, indicating that the drug composition of the present invention can effectively treat PEDV-induced diarrhea in pigs, relieve diarrhea symptoms, and the therapeutic effect is significantly better than that of single-component drugs. Moreover, the therapeutic effects of different dosage forms are relatively stable and suitable for clinical promotion and use.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition for treating swine diarrhea, characterized in that, It includes the following raw materials: spiculin and Wangbuliuxing cyclic peptide B, wherein the mass ratio of spiculin to Wangbuliuxing cyclic peptide B is 1:(0.15-0.25).

2. The pharmaceutical composition for treating swine diarrhea according to claim 1, characterized in that, The mass ratio of strychnine and Wangbuliuxing cyclic peptide B is 1:0.

2.

3. The pharmaceutical composition for treating swine diarrhea according to claim 1, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The pharmaceutical composition for treating swine diarrhea according to claim 3, characterized in that, The dosage form of the pharmaceutical composition is any one of tablets, powders, granules, oral liquids, and injections.

5. A method for preparing a pharmaceutical composition for treating swine diarrhea according to any one of claims 1-4, characterized in that, Includes the following steps: According to the formula, mix the cystic acid and Wangbuliuxing cyclic peptide evenly, and add pharmaceutically acceptable excipients to obtain the final product.

6. The method for preparing a pharmaceutical composition for treating swine diarrhea according to claim 5, characterized in that, The pharmaceutically acceptable excipients include at least one of lactose, soluble starch, and wheat bran.

Images