Application of pithecellobium clypearia extract in preparation of anti-porcine reproductive and respiratory syndrome medicine

Abstract

The invention discloses application of pithecellobium clypearia extract in preparation of anti-porcine reproductive and respiratory syndrome drugs. The pithecellobium clypearia is found to be capable of remarkably inhibiting infection of porcine reproductive and respiratory syndrome virus (PRRSV) on a susceptible cell Marc-145 and inhibiting proliferation of the PRRSV for the first time, and is expected to be used for preparing the medicine for preventing and treating the porcine reproductive and respiratory syndrome. The pithecellobium clypearia extract disclosed by the invention not only provides a new medicine source for preventing and treating the porcine reproductive and respiratory syndrome, but also widens the application range of the pithecellobium clypearia, and has a wide application prospect for preventing and treating the porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] The 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 is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). The Ministry of Agriculture and Rural Affairs has classified highly pathogenic blue ear disease as a Class I animal epidemic disease. The clinical symptoms of host animal pigs infected with PRRSV include abortion, premature birth, stillbirth, weak or mummified fetuses in late pregnancy, anorexia, fever, and respiratory disorders in sick pigs of all ages (especially piglets). At the same time, PRRSV infection leads to low host immune function, increased susceptibility to other pathogenic factors, and mixed infection with multiple bacteria, which has brought huge economic losses to the global pig industry.

[0003] At present, the main means of preventing and controlling blue ear disease in farms is vaccines. Vaccines have the risk of spreading viruses, which may cause incomplete humoral immunity and lead to long-term persistent infection in pig farms. In the face of the outbreak of blue ear disease, there is no specific drug to prevent and treat PRRSV, and antibiotics can only be used to treat secondary bacterial infections caused by the virus. Excessive use of antibiotics can cause animals to develop serious drug resistance, and irrational use of drugs can also cause a large amount of veterinary drugs to remain in the body of animals, which in turn has adverse effects on human health.

[0004] Traditional Chinese medicine has the advantages of rich resources, low residue, no drug resistance and low toxicity and side effects. Using traditional Chinese medicine in pig feed to prevent and treat blue ear disease can not only reduce costs, prevent diseases, improve production performance, but also improve the quality of livestock and poultry products. Monkey earrings are the dried young branches and leaves of the leguminous plant Archidendron clypearia (Jack.) Nielsen, which is mainly distributed 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 monkey earring extracts have an inhibitory effect on influenza virus neuraminidase (NA). The compounds isolated from monkey earrings have good anti-respiratory syncytial virus, influenza virus H1N1, Coxsackie virus B3 and herpes simplex virus type I activity, but there is no report on the anti-PRRSV activity of monkey earrings. Summary of the invention

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

[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 the use of monkey earring extract in preparing 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 porcine blue ear disease.

[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 earrings are extracted with 95% ethanol aqueous solution, filtered, and the filtrate is concentrated and dried to obtain an extract. The extract is re-dissolved in water and extracted with petroleum ether, ethyl acetate, and n-butanol, leaving the aqueous phase to obtain a petroleum ether ether fraction, an ethyl acetate fraction, an n-butanol fraction and an aqueous phase, respectively, which are concentrated and dried, thereby obtaining the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and aqueous phase fraction 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 solution.

[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 PRRSV recombinant virus strain carrying fluorescent EGFP to infect cells, and under a fluorescent microscope, it is 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 type of biologically active substance for preventing and treating 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 earrings, 95% ethanol extract of monkey earrings, petroleum ether extraction part of 95% ethanol extract of monkey earrings, ethyl acetate extraction part of 95% ethanol extract of monkey earrings, n-butanol extraction part of 95% ethanol extract of monkey earrings, and aqueous extraction part of 95% ethanol extract of monkey earrings. DETAILED DESCRIPTION

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

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

[0023] The monkey earrings (HEH) used in the embodiments 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 are both stored in the Animal Genetics and Breeding Laboratory of the School of Life Sciences of Sun Yat-sen University; the MTS cell activity detection kit is purchased from Promega Company.

[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 identified by Chief Pharmacist Peng Wei of Sun Yat-sen University) were weighed and put into a 500 mL round-bottom flask, and 300 mL of 95% ethanol was poured in to immerse the medicinal materials overnight. The residue was filtered, the medicinal solution was concentrated, and the solution was placed in a freeze dryer for freeze drying overnight to obtain HEH alcohol extract (HEH-alcohol extract, i.e., monkey earring 95% ethanol extract).

[0027] 1.645g of 95% ethanol extract of monkey earrings was re-dissolved with 200mL water, fully shaken and mixed, poured into a 500mL separatory funnel, and placed on an iron stand. 200mL petroleum ether was first poured in and fully shaken, and the upper petroleum ether layer was recovered and extracted once; the remaining aqueous phase was then repeatedly extracted with 200mL ethyl acetate and 200mL n-butanol in sequence, leaving the aqueous phase, and the obtained petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and aqueous phase were respectively concentrated, placed in a freeze dryer and freeze-dried overnight, thereby obtaining the extraction fractions of the monkey earring extract, namely, 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 for freeze drying overnight, weigh it the next day, and get 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 incubated at 5×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate and placed in a 37°C, 5% CO2 cell culture incubator for overnight culture to allow them to adhere to the wall and grow. The control group was added with a cell maintenance solution containing DMSO (DMEM containing 2% FBS), and the drug-treated group was added with different concentrations of the monkey earring extracts to be tested or different extraction parts or ribavirin and cultured in a 37°C, 5% CO2 cell culture incubator for 72 hours. Only PBS was added as the background control well, and 100 μL of DMEM culture medium containing 2% FBS and 100 μL of virus solution were added to each well as the virus control group, and only 200 μL of DMEM culture medium containing 2% FBS was added as the cell control group. Each group had 3 replicate wells. The cell lesions and fluorescence intensity were observed under a fluorescence microscope every day, and the cell lesions in each well were recorded and photographed. The old culture medium was discarded, and fresh culture medium containing 10% MTS was added to continue culturing for 1 to 2 hours. The OD value was measured, and the half-maximal inhibitory concentration (IC) 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 The cells were inoculated at a density of 100 / mL in a 96-well plate and placed in a 37°C, 5% CO2 cell culture incubator for overnight culture to allow them to adhere to the wall and grow. The control group was added with a cell maintenance medium containing DMSO (DMEM containing 2% FBS), and the drug-treated group was added with different concentrations of the monkey earring extracts to be tested, different extraction parts and ribavirin in a 37°C, 5% CO2 cell culture incubator for 72 hours. Only PBS was added as a background control well, and a cell control group (cell maintenance medium without compounds) was set up at the same time. Each group had 3 replicate wells. Continue to culture for 72 hours, discard the old culture medium, add fresh culture medium containing 10% MTS and continue to culture for 1 to 2 hours, measure the OD value, and calculate the half lethal concentration (CC) of the compound using Graphpad Prime 6.0 software. 50 ).

[0033] Firstly, the virus strain carrying PRRSV-EGFP was used to screen whether monkey earring water extract, monkey earring water extract (HEH-water extract), monkey earring 95% ethanol extract (HEH-alcohol 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), monkey earring 95% ethanol extract aqueous extract (HEH-Water) had anti-PRRSV activity. The results were as follows: Figure 2-Figure 7 MTS was used to detect the CC of the above samples on Marc-145 cells. 50 ; MTS combined with CPE method was used to detect the IC of the above samples against PRRSV-EGFP virus strain 50 The specific results are shown in Table 1. The results showed that the monkey earring extracts all had an inhibitory effect on PRRSV-EGFP. It is worth noting that the IC values ​​of the monkey earring water extract, the monkey earring 95% ethanol extract, and the monkey earring ethyl acetate fraction on PRRSV 50 The IC value of monkey earring 95% ethanol extract was less than that of the positive control drug ribavirin. 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. Application of monkey earring extract in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs or feed additives.

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

3. The use according to claim 1 or 2, characterized in that: The monkey earring extract is an extract of water, alcohol or alcohol aqueous solution of monkey earring.

4. The use according to claim 3, characterized in that: The alcohol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

5. The use according to claim 1 or 2, characterized in that: The monkey earring extract is obtained by leaching the monkey earring with water or 95% ethanol aqueous solution, filtering, concentrating and drying the filtrate.

6. The use according to claim 1 or 2, characterized in that: The monkey earrings are extracted with 95% ethanol aqueous solution, filtered, and the filtrate is concentrated and dried to obtain an extract. The extract is re-dissolved in water and extracted with petroleum ether, ethyl acetate, and n-butanol, leaving the aqueous phase to obtain a petroleum ether ether portion, an ethyl acetate portion, an n-butanol portion, and an aqueous phase, respectively, which are concentrated and dried, thereby obtaining the petroleum ether ether portion, ethyl acetate portion, n-butanol portion, and aqueous phase portion of the monkey earring extract, namely, each monkey earring extract.

7. An anti-porcine reproductive and respiratory syndrome virus drug, characterized in that: The drug contains monkey earring extract as an active ingredient, and preferably, the drug dosage form is powder, oral solution or injection solution.

8. The anti-porcine reproductive and respiratory syndrome virus drug according to claim 7, characterized in that: The monkey earring extract is an extract of water, alcohol or alcohol aqueous solution of monkey earring. Preferably, the alcohol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

9. The anti-porcine reproductive and respiratory syndrome virus drug according to claim 7, characterized in that: The monkey earring extract is obtained by leaching the monkey earring with water or 95% ethanol aqueous solution, filtering, concentrating and drying the filtrate.

10. The use according to claim 7, characterized in that: The monkey earring extract is prepared by leaching the monkey earring with a 95% ethanol aqueous solution, filtering, concentrating and drying the filtrate to obtain an extract, re-dissolving the extract with water, extracting with petroleum ether, ethyl acetate and n-butanol, leaving the aqueous phase, respectively obtaining a petroleum ether ether portion, an ethyl acetate portion, an n-butanol portion and an aqueous phase, respectively concentrating and drying, thereby obtaining a petroleum ether ether portion, an ethyl acetate portion, an n-butanol portion and an aqueous phase portion of the monkey earring extract, namely each monkey earring extract.

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