Application of monkey earring extract in preparation of anti-blue ear disease drugs

Abstract

The present invention discloses the use of monkey earring extract in the preparation of anti-blue ear disease drugs. The present invention is the first to study and find that monkey earring can significantly inhibit the infection of porcine reproductive and respiratory syndrome virus (PRRSV) in its susceptible cell Marc-145, inhibiting the proliferation of PRRSV, and is expected to be used in the preparation of drugs for the prevention and treatment of blue ear disease. The present invention not only provides a new drug source for the prevention and treatment of blue ear disease in pigs, but also broadens the scope of application of monkey earring, and has broad application prospects for the prevention and treatment of blue ear disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal husbandry medicine, and more specifically relates to the application of monkey earring extract in the preparation of anti-blue ear disease medicine. Background Art

[0002] Blue ear disease (PRRS) is a highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The Ministry of Agriculture and Rural Affairs classifies highly pathogenic PRRS as a Class I animal disease. Clinical symptoms in pigs infected with PRRSV include miscarriage, premature birth, stillbirth, weak or mummified fetuses in late gestation, and anorexia, fever, and respiratory impairment in pigs of all ages (especially piglets). Furthermore, PRRSV infection weakens the host immune system, increasing susceptibility to other pathogens and leading to mixed infections with multiple bacteria, resulting in significant economic losses for the global pig industry.

[0003] Currently, the primary means of preventing and controlling blue ear disease (PRRS) on farms is through vaccines. However, these vaccines carry the risk of viral shedding, potentially leading to incomplete humoral immunity and persistent long-term infection in pig farms. In the face of outbreaks of PRRSV, there is no specific medication to prevent or treat PRRSV, leaving only antibiotics to treat secondary bacterial infections caused by the virus. Excessive antibiotic use can lead to severe drug resistance in animals, while inappropriate use can also result in high levels of veterinary drug residues in animals, which can negatively impact human health.

[0004] Traditional Chinese medicine (TCM) boasts advantages such as abundant resources, low residue levels, resistance to drug resistance, and minimal toxic side effects. Using TCM in pig feed to prevent and treat blue ear disease not only reduces costs, prevents disease, and improves production performance, but also improves the quality of livestock and poultry products. Archidendron clypearia (Jack.) Nielsen, a plant of the Leguminosae family, is a dried young branch and leaf of the genus Archidendron clypearia (Jack.), primarily found in Guangdong and Guangxi. It has the effects of clearing heat and detoxifying, cooling blood and reducing swelling, and stopping diarrhea. Studies have reported that extracts from Archidendron clypearia inhibit influenza virus neuraminidase (NA). Compounds isolated from Archidendron clypearia have demonstrated significant activity against respiratory syncytial virus, influenza virus H1N1, coxsackievirus B3, and herpes simplex virus type 1. However, the anti-PRRSV activity of Archidendron clypearia has not been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing technologies and provide an application of monkey earring extract in the preparation of anti-blue ear disease drugs.

[0006] The purpose of the invention is to develop new uses of monkey earrings in the pharmaceutical field.

[0007] Therefore, the first object of the present invention is to provide a use of the monkey earring extract in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs or feed additives.

[0008] Preferably, the invention is used in the preparation of a drug for treating blue ear disease in pigs.

[0009] Preferably, the monkey earring extract is an extract of monkey earring in water, alcohol or alcohol-water solution.

[0010] Preferably, the alcohol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

[0011] Preferably, the monkey earrings are extracted with water or 95% ethanol aqueous solution, filtered, and the filtrate is concentrated and dried to obtain the product.

[0012] Further preferably, the monkey earring is extracted with 95% ethanol aqueous solution, filtered, and the filtrate is concentrated and dried to obtain an extract. The extract is redissolved with water and extracted with petroleum ether, ethyl acetate, and n-butanol, leaving the aqueous phase to obtain the petroleum ether portion, ethyl acetate portion, n-butanol portion and aqueous phase, respectively, which are concentrated and dried, thereby obtaining the petroleum ether portion, ethyl acetate portion, n-butanol portion and aqueous phase portion of the monkey earring extract, namely, each monkey earring extract.

[0013] The second object of the present invention is to provide an anti-porcine reproductive and respiratory syndrome virus drug, characterized in that it contains monkey earring extract as an active ingredient.

[0014] As a preferred embodiment, the medicine contains a therapeutically effective amount of monkey earring extract and a pharmaceutically acceptable carrier.

[0015] The dosage form of the drug can be prepared into different desired dosage forms, such as powder, oral solution or injection.

[0016] The present invention has the following beneficial effects:

[0017] (1) The present invention is the first to discover that the monkey earring extract has significant anti-PRRSV activity and can be used as a natural antiviral drug for the prevention and treatment of blue ear disease in pigs. At the same time, it can achieve the comprehensive utilization of the monkey earring plant and increase its added value, and has a good application prospect.

[0018] (2) Specifically, the present invention uses a recombinant PRRSV virus strain carrying fluorescent EGFP to infect cells. Under a fluorescence microscope, it was observed that the monkey earring extract can significantly inhibit PPRSV infection and replication, has a significant antiviral effect, and is expected to become a new bioactive substance for the prevention and treatment of blue ear disease. The product prepared by this method. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1It is the process flow of each extraction part of monkey earring 95% ethanol extract;

[0020] Figure 2-7 The anti-PRRSV activity results are respectively the water extract of monkey earring, 95% ethanol extract of monkey earring, petroleum ether extraction part of 95% ethanol extract of monkey earring, ethyl acetate extraction part of 95% ethanol extract of monkey earring, n-butanol extraction part of 95% ethanol extract of monkey earring, and aqueous extraction part of 95% ethanol extract of monkey earring. DETAILED DESCRIPTION

[0021] The present invention is further described below with reference to specific examples, which do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are those commonly used in the art.

[0022] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0023] The monkey earrings (HEH) used in the examples of the present invention were purchased from Guangzhou Caizhilin Pharmaceutical Co., Ltd.

[0024] The porcine reproductive and respiratory syndrome virus PRRSV EGFP recombinant virus strain and Marc-145 (African green monkey embryonic kidney cells) used in the present invention were both preserved in the Animal Genetics and Breeding Laboratory of the School of Life Sciences of Sun Yat-sen University; the MTS cell activity detection kit was purchased from Promega.

[0025] Example 1: Preparation of 95% ethanol extract of monkey earrings and its effective fraction

[0026] like Figure 1 As shown, 30 g of monkey earring medicinal materials (purchased from Guangzhou Caizhilin Pharmaceutical Co., Ltd. and authenticated by Chief Pharmacist Peng Wei of Sun Yat-sen University) were weighed and placed in a 500 mL round-bottom flask. 300 mL of 95% ethanol was poured into the flask to submerge the medicinal materials overnight. The residue was filtered, the liquid medicine was concentrated, and the mixture was freeze-dried in a freeze dryer overnight to obtain HEH alcohol extract (HEH-alcohol extract, i.e., 95% ethanol extract of monkey earring).

[0027] 1.645 g of the 95% ethanol extract of monkey earrings was reconstituted with 200 mL of water, thoroughly shaken and mixed, and poured into a 500 mL separatory funnel, which was then placed on an iron stand. 200 mL of petroleum ether was first poured in, shaken thoroughly, and allowed to stand for stratification. The upper petroleum ether layer was recovered and extracted once again; the remaining aqueous phase was then repeatedly extracted with 200 mL of ethyl acetate and 200 mL of n-butanol, leaving the aqueous phase. The resulting petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and aqueous phase were concentrated separately and freeze-dried overnight in a freeze dryer to obtain the extracted fractions of the monkey earring extract, namely the petroleum ether fraction (HEH-PE), ethyl acetate fraction (HEH-EA), n-butanol fraction (HEH-n-butanol), and aqueous phase (HEH-Water).

[0028] Example 2: Preparation of the effective fraction of monkey earring water extract

[0029] Weigh 30 g of monkey earring medicinal materials, put them into a 500 mL round-bottom flask, pour in 300 mL of water, immerse the medicinal materials for half an hour, use an electric heating mantle to heat until boiling, stop heating after 1 hour, filter the medicinal residue after cooling, concentrate the medicinal liquid, put it into a freeze dryer and freeze-dry overnight, weigh it the next day, and obtain 178.7 mg of monkey earring water extract (HEH-water extract), with an extraction rate of 0.6%.

[0030] Example 3: In vitro anti-PRRSV activity test

[0031] The cytopathic effect (CPE) method combined with the MTS method was used to detect the activity of monkey earring extract and different extraction parts against PRRSV: Marc-145 cells were plated at 5×10 4 Cells were seeded at a density of 100 μL / mL in a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell culture incubator to allow them to adhere to the wall. The control group was treated with DMSO-containing cell maintenance medium (DMEM containing 2% FBS), and the drug-treated group was treated with different concentrations of the monkey earring extract or different extraction parts or ribavirin and cultured in a 37°C, 5% CO2 cell culture incubator for 72 hours. PBS alone was added as the background control well. 100 μL of DMEM culture medium containing 2% FBS and 100 μL of virus solution were added to each well as the virus control well. Only 200 μL of DMEM culture medium containing 2% FBS was added to the cell control well. Three replicate wells were set up for each group. The cytopathic effect and fluorescence intensity were observed under a fluorescence microscope every day, and the cytopathic effect of each well was recorded and photographed. The old culture medium was discarded, and fresh culture medium containing 10% MTS was added to continue culturing for 1-2 hours. The OD value was measured, and the half-maximal inhibitory concentration (IC50) of the compound was calculated using Graphpad Prime 6.0 software. 50 ), the results are shown in Table 1.

[0032] MTS method was used to detect the cytotoxicity of monkey earring extract and different extraction parts and ribavirin on Marc-145 cells: Marc-145 cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / mL in a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell culture incubator to allow them to adhere to the wall. The control group was treated with DMSO-containing cell maintenance medium (DMEM containing 2% FBS). The drug-treated group was treated with different concentrations of the monkey earring extract to be tested, different extraction parts, and ribavirin and cultured in a 37°C, 5% CO2 cell culture incubator for 72 hours. PBS alone was added as a background control well. A cell control group (cell maintenance medium without compound) was also set up. Three replicate wells were set up for each group. After 72 hours of culture, the old culture medium was discarded and fresh culture medium containing 10% MTS was added to continue culture for 1-2 hours. The OD value was measured and the half-lethal concentration (CC) of the compound was calculated using Graphpad Prime 6.0 software. 50 ).

[0033] Firstly, a virus strain carrying PRRSV-EGFP was used to screen whether monkey earring water extract (HEH-water extract), monkey earring 95% ethanol extract (HEH-ethanol extract), monkey earring 95% ethanol extract petroleum ether extract (HEH-PE), monkey earring 95% ethanol extract ethyl acetate extract (HEH-EA), monkey earring 95% ethanol extract n-butanol extract (HEH-n-butanol), and monkey earring 95% ethanol extract aqueous extract (HEH-Water) had anti-PRRSV activity. Figure 2-Figure 7 MTS was used to detect the CC of Marc-145 cells by the above samples. 50 The IC values of the above samples for PRRSV-EGFP virus strain were detected by MTS combined with CPE method. 50 The specific results are shown in Table 1. The results showed that the monkey earring extracts all had inhibitory effects on PRRSV-EGFP. It is worth noting that the IC values of monkey earring water extract, monkey earring 95% ethanol extract, and monkey earring ethyl acetate fraction on PRRSV were 50 Less than the positive control drug ribavirin, among which the IC of 95% ethanol extract of monkey earrings 50 The value is the smallest.

[0034] Table 1. Anti-porcine reproductive and respiratory syndrome virus activity test results of monkey earring extract and its extraction parts

[0035]

Claims

1. Use of monkey earring extract in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs, wherein the monkey earring extract is an extract of monkey earring water and alcohol aqueous solution, and the alcohol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

2. The use according to claim 1, characterized in that The monkey earring extract is the ethyl acetate extraction portion of the monkey earring alcohol aqueous solution extract.

3. The use according to claim 1, characterized in that The invention is used in preparing medicine for treating porcine blue ear disease.

4. The use according to claim 1 or 3, characterized in that The monkey earring water and alcohol aqueous solution extract is obtained by leaching the monkey earring with water or 95% ethanol aqueous solution, filtering, and concentrating and drying the filtrate.

5. The use according to claim 2, characterized in that The monkey earrings were extracted with a 95% ethanol aqueous solution, filtered, and the filtrate was concentrated and dried to obtain an extract. The extract was redissolved with water and extracted with petroleum ether, ethyl acetate, and n-butanol, leaving the aqueous phase to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction, and an aqueous phase, respectively. The fractions were concentrated and dried to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and aqueous phase fraction of the monkey earring extract. The ethyl acetate fraction was taken as the ethyl acetate extraction fraction of the extract of the alcohol aqueous solution.

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