EHPA injection preparation for treating porcine viral diarrhea

Abstract

According to the EHPA injection preparation for treating porcine viral diarrhea, Euphorbia hupehensis phenolic acid compounds (EHPA) are separated from roots of Euphorbia hupehensis for the first time and prepared into the injection preparation, after optimization, the stability of the injection preparation is high, the quality and characters of products within the shelf life are stable, the EHPA injection preparation is safe to animals, and animal experiments show that the EHPA injection preparation has good clinical application prospects. The compound has a good control effect on diarrhea caused by PEDV and / or PDCoV infection, can effectively reduce death of infected animals, and has an excellent treatment effect.

Description

Technical fields:

[0001] This invention belongs to the field of biotechnology and relates to the treatment of swine viral diarrhea, specifically to an injectable preparation of EHPA, a phenolic acid compound from Hubei Province, and its application in the treatment of swine viral diarrhea. Background technology:

[0002] Porcine viral diarrhea (PDD) is a common digestive tract disease caused by porcine enterovirus infection, seriously threatening the healthy development of the pig farming industry. Multiple porcine enteroviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine ridge virus (PKV), can all cause PDD. In clinical practice, mixed infections of multiple viruses can exacerbate intestinal pathological damage and lead to continuous viral mutations, challenging the immunoprotective efficacy of vaccines and highlighting the urgent need for drugs to treat related infections.

[0003] *Euphorbia hylonoma*, commonly known as Hubei Euphorbia, is a perennial herb belonging to the genus *Euphorbia* in the family Euphorbiaceae. The entire plant is smooth and hairless, growing in mountain valleys, slopes, thickets, grasslands, and sparse forests at altitudes of 200-3000 meters. It has a relatively large population. There are approximately 2000 species of *Euphorbia* worldwide. These plants contain white or yellowish-white latex, and their medicinal value is well-known due to the presence of some physiologically active components. Traditional Chinese medicines such as *Euphorbia hylonoma*, *Euphorbia kansui*, and *Euphorbia helioscopia* are recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). *Euphorbia* plants mainly contain diterpenoid esters, triterpenoids, sterols, flavonoids, tannins, and phenolic compounds. A notable characteristic of this genus is the presence of a white or yellowish-white latex, which is irritating to the skin and is primarily used in traditional medicine for wart removal and anti-tumor purposes.

[0004] Currently, numerous studies have been conducted on the secondary metabolites and related activities of *Euphorbia hylonoma*. Among them, Chinese patent application CN200810018230.5 discloses an ellagic acid compound isolated from the roots of *Euphorbia hylonoma*, which exhibits anticancer activity; Chinese patent application CN201810394704.X discloses an enantiopinene-type diterpenoid compound with herbicidal activity; Li Juan et al. (“Cyclo-Altin-type Triterpenoids in the Roots of *Euphorbia hylonoma*”, *Natural Product Research and Development*, Vol. 19, 2007, pp. 995-997) obtained *Euphorbia hylonoma* from the Shennongjia region of Hubei Province. Four cycloaltin-type triterpenoids were isolated for the first time from the roots of *Hand.-Mazz.* and identified as: 24-enylcycloaltin palmitate (1), cycloaltin-23-en-3β,25-diol-3-acetate (2), cycloaltin-25-en-3β,24ξ-diol (3), and cycloaltin-23-en-3β,25,28-triol (4). Ruan Hanli et al. (“Research on Chemical Constituents of *Euphorbia hylonoma*, *Chinese Journal of Traditional Chinese Medicine*, Vol. 31, No. 9, 2006, pp. 742-744) studied the chemical constituents of the roots of *Euphorbia hylonoma* and isolated and identified six compounds for the first time: n-nonane (1), bis(2-ethylhexyl)phthalate (2), euphorbia alcohol (3), β-sitosterol (4), C50 long-chain fatty alcohol (5), and β-carotene (6). Ruan Hanli et al. (“Tanoids in Euphorbia hupehensis”, Chinese Traditional and Herbal Drugs, Vol. 37, No. 12, 2006, pp. 1782-1784) studied Euphorbia hupehensis produced in the Shennongjia area of ​​Hubei Province. They obtained seven components from the ethyl acetate fraction, which were identified as 3,3′,4′-trimethylellagic acid (Ⅰ), 4-methyl-gallic acid (Ⅱ), gallic acid (Ⅲ), 3,3′-dimethylellagic acid-4′-O-β-D-xylanoside (Ⅳ), 3,3′-dimethylellagic acid (Ⅴ), 1,2,3,4,6-pentaoxaloyl-β-D-glucose (Ⅵ), and sucrose (Ⅶ). Compounds Ⅱ to Ⅵ were isolated from this plant for the first time. Based on this, the inventors, after years of research, isolated a phenolic acid compound, EHPA, from the roots of Euphorbia hupehensis and experimentally verified its antiviral activity. However, how to apply EHPA to the preparation of specific drugs and the treatment of diseases is also a technical problem that needs to be solved. Summary of the Invention:

[0005] The present invention aims to provide an injectable formulation of euphorbia phenolic acid compound EHPA, which can maintain the stability of active ingredients over a long storage period, has a long half-life, and low drug residues. Based on the inventors' previous research, the present invention aims to use this injectable formulation for the treatment of diarrheal diseases caused by viral infections.

[0006] An injectable formulation of euphorbia pekinensis phenolic acid compound EHPA, wherein each 1000mL injection formulation comprises 0.2-0.4g of euphorbia pekinensis phenolic acid compound EHPA, 10-20g of astragalus polysaccharide, 50-70g of complex organic solvent, 2-4g of surfactant, 5-10g of buffer salt composed of NaH2PO4 and Na2HPO4, 20g of NaCl and 1-2g of antioxidant.

[0007] The chemical structural formula of the phenolic acid compound EHPA from Hubei is shown in formula (I):

[0008]

[0009] The preparation method of the phenolic acid compound EHPA from Hubei Euphorbia is as follows:

[0010] (1) Ethanol extraction: The roots of the naturally dried Euphorbia hupehensis were crushed using a pulverizer. The crushed powder was then added to ethanol for extraction. After filtration, the filtrate was concentrated under reduced pressure to obtain an ethanol extract.

[0011] (2) n-Butanol extraction: The extract obtained in step (1) is suspended in water and then extracted with petroleum ether, ethyl acetate and n-butanol in sequence. The n-butanol extract is concentrated under reduced pressure to obtain n-butanol extract.

[0012] (3) First column chromatography: The n-butanol extract obtained in step (2) is separated by column chromatography. The packing material is 200-300 mesh silica gel. Chloroform-methanol with a volume ratio of 15 / 1 to 1 / 4 is used as the eluent for gradient elution. The eluent containing the target compound is collected. The solvent is removed by vacuum distillation to obtain the first column chromatography product.

[0013] (4) Secondary column chromatography: The primary column chromatography product obtained in step (3) is further separated by column chromatography. The packing material is 200-300 mesh silica gel. Chloroform-methanol with a volume ratio of 100:1 to 1:1 is used as the eluent for gradient elution. The eluent containing the target compound is collected. The solvent is removed by vacuum distillation to obtain the secondary column chromatography product.

[0014] (5) Preparation and purification: The secondary column chromatography product obtained in step (4) is separated again by a gel column LH-20, and then purified by semi-preparative HPLC to obtain the compound shown in formula (I).

[0015] Preferably, the specific operation method of step (1) is as follows: the dried and pulverized Euphorbia pekinensis root powder is extracted with ethanol, the mass-volume ratio between Euphorbia pekinensis root powder and ethanol is 1 / 5 to 1 / 10 (kg / L), the extraction is carried out at room temperature for 5 to 10 days, and then the ethanol extract is obtained by filtration. The filter residue is extracted 2 to 4 times, the ethanol extracts obtained each time are combined, and the solvent is recovered by vacuum concentration to obtain ethanol extract.

[0016] Preferably, the concentration of ethanol, the extraction solvent used in step (1), is 95%.

[0017] Preferably, in step (2), the n-butanol is extracted 2-4 times, preferably 3 times. The n-butanol extract phases are combined, and the solvent is recovered under reduced pressure to obtain n-butanol extract.

[0018] Preferably, the eluent for the gel column LH-20 in step (5) is 50% methanol.

[0019] Preferably, the mobile phase of the semi-preparative HPLC in step (5) is 30% methanol.

[0020] Preferably, the Hubei Euphorbia pekinensis phenolic acid compound EHPA injection formulation comprises 0.4g Hubei Euphorbia pekinensis phenolic acid compound EHPA, 15g Astragalus polysaccharide, 60g compound organic solvent, 3g surfactant, 8g buffer salt composed of NaH2PO4 and Na2HPO4, 20g NaCl and 2g antioxidant per 1000mL of injection formulation.

[0021] Preferably, the composite organic solvent is composed of propylene glycol, dimethylformamide, or ethanol. More preferably, the composite organic solvent is prepared by mixing ethanol and propylene glycol in a volume ratio of 2:1.

[0022] Preferably, the surfactant is selected from one or both of Span-80 and Tween-20; more preferably, the surfactant is composed of Span-80 and Tween-20 in a mass ratio of 2:1.

[0023] Preferably, the antioxidant is selected from sodium bisulfite, sodium pyrosulfite and / or disodium EDTA. More preferably, the antioxidant is composed of sodium pyrosulfite and EDTA-2Na in a mass ratio of 2:1.

[0024] More preferably, the Hubei Euphorbia pekinensis phenolic acid compound EHPA injection formulation comprises, per 1000 mL, 0.4 g Hubei Euphorbia pekinensis phenolic acid compound EHPA, 15 g Astragalus polysaccharide, 60 g composite organic solvent, 3 g surfactant, 8 g buffer salt composed of NaH2PO4 and Na2HPO4, 20 g NaCl, and 2 g antioxidant. The composite organic solvent is prepared by mixing ethanol and propylene glycol in a volume ratio of 2:1. The surfactant is composed of Span-80 and Tween-20 in a mass ratio of 2:1. The antioxidant is composed of sodium pyrosulfate and EDTA-2Na in a mass ratio of 2:1.

[0025] This invention also claims protection for a method for preparing the injectable formulation of the phenolic acid compound EHPA from Hubei Province, comprising the following steps:

[0026] 1) Weigh the raw materials according to the following ratio for each 1000mL injection preparation: 0.2-0.4g of euphorbia pekinensis phenolic acid compound EHPA, 10-20g of astragalus polysaccharide, 50-70g of compound organic solvent, 2-4g of surfactant, 5-10g of buffer salt composed of NaH2PO4 and Na2HPO4, 20g of NaCl, 1-2g of antioxidant and the balance of water for injection.

[0027] 2) Take 30% of the total volume of the injection solution and heat it to 55-60℃. Then add the weighed Astragalus polysaccharide, the buffer salt composed of NaH2PO4 and Na2HPO4 and NaCl, and stir until it is completely dissolved to obtain solution A.

[0028] 3) Take 50% of the total volume of the injection solution and heat it to 40-45℃ for later use; heat the weighed compound organic solvent to 40-45℃, then add the weighed Hubei Euphorbia pekinensis phenolic acid compound EHPA and stir evenly, then add the water for injection that has been heated to 40-45℃, the weighed surfactant and antioxidant and stir until completely dissolved to obtain solution B.

[0029] 4) Slowly add solution A to solution B, then add the remaining water for injection, stir well, fill and sterilize to obtain the Hubei Euphorbia phenolic acid compound EHPA injection preparation.

[0030] Preferably, the preparation method of the Hubei Euphorbia pekinensis phenolic acid compound EHPA injection includes the following steps:

[0031] 1) Weigh the raw materials according to the following ratio per 1000mL injection preparation: 0.4g of Hubei Euphorbia pekinensis phenolic acid compound EHPA, 15g of Astragalus polysaccharide, 60g of complex organic solvent, 3g of surfactant, 8g of buffer salt composed of NaH2PO4 and Na2HPO4, 20g of NaCl, 2g of antioxidant, and the balance being water for injection. The complex organic solvent is prepared by mixing ethanol and propylene glycol in a volume ratio of 2:1. The surfactant is composed of Span-80 and Tween-20 in a mass ratio of 2:1. The antioxidant is composed of sodium pyrosulfate and EDTA-2Na in a mass ratio of 2:1.

[0032] 2) Take 30% of the total volume of the injection solution and heat it to 55°C. Then add the weighed Astragalus polysaccharide, the buffer salt composed of NaH2PO4 and Na2HPO4 and NaCl, and stir until it is completely dissolved to obtain solution A.

[0033] 3) Take 50% of the total volume of the injection solution and heat the water for injection to 42°C for later use; heat the weighed compound organic solvent to 42°C, then add the weighed Hubei Euphorbia pekinensis phenolic acid compound EHPA and stir evenly, then add the water for injection that has been heated to 42°C, the weighed surfactant and antioxidant and stir to completely dissolve to obtain solution B.

[0034] 4) Slowly add solution A to solution B, then add the remaining water for injection, stir well, fill and sterilize to obtain the Hubei Euphorbia phenolic acid compound EHPA injection preparation.

[0035] The EHPA injection formulation of phenolic acid compounds from Hubei Province prepared using the above method is a clear yellow liquid.

[0036] Based on the above technical solution, the present invention has the following beneficial effects:

[0037] First, the inventors isolated the phenolic acid compound (EHPA) (the compound of formula I) from the root of Euphorbia hubeiensis for the first time. The isolation and purification method is simple. The compound was found to be highly safe and non-toxic to cells and animals. Furthermore, the activity test revealed that the compound has antiviral activity and has an inhibitory effect on viruses such as porcine epidemic diarrhea virus (PEDV) and porcine PDCoV that cause diarrhea in pigs.

[0038] Secondly, the injectable formulation of this invention exhibits high stability after optimization, with consistent quality and properties throughout its shelf life, and is safe for animals. Furthermore, animal studies have shown that it effectively controls diarrhea caused by PEDV and / or PDCoV infection, significantly reducing mortality in infected animals and demonstrating excellent therapeutic efficacy. Detailed implementation method:

[0039] The following embodiments are descriptions of preferred embodiments of the present invention. It should be understood that the embodiments described herein are for illustration and explanation only, and are not intended to limit the present invention.

[0040] Example 1. Preparation method of euphorbia pekinensis phenolic acid compound (EHPA) from Hubei

[0041] 10 kg of naturally dried roots of Euphorbia pekinensis from Hubei Province were pulverized using a pulverizer. The pulverized powder was then extracted by soaking in 80 L of 95% ethanol at room temperature for 5 days each time, for a total of 3 soakings. After filtration, the filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a temperature below 45°C to obtain a total extract of 2.2 kg.

[0042] The total extract was suspended in 2000 ml of distilled water and then extracted sequentially with petroleum ether (2000 ml × 3), ethyl acetate (2000 ml × 3), and n-butanol (2000 ml × 3). The extracts with the same extraction solvent were combined, and the n-butanol extract was concentrated under reduced pressure to recover the solvent, yielding 285 g of extract. The extract was first subjected to column chromatography with 200-300 mesh silica gel for rough separation, and gradient elution was performed with chloroform-methanol (v / v, 15:1 to 1:4) as the eluent to obtain 8 fractions Fr.1-Fr.8.

[0043] Fr.5 (55.6 g) was further fractionated on a 200–300 mesh silica gel column using a gradient elution of chloroform-methanol (v / v, 100:1 to 1:1) to obtain three fractions (Fr.5.1 to Fr.5.3). Fr.5.2 (13 g) was separated by dextran gel LH-20 (solvent: 50% methanol) and further purified by semi-preparative HPLC (30% methanol aqueous solution) to obtain 52 mg of compound (I).

[0044] Analysis revealed that the molecular formula of the compound is C2. 19 H 20 O8 is a white solid powder with a melting point of 75-77℃. The 1H and 13C NMR (DMSO-D6) spectra of compound (I) are shown in Table 1 below:

[0045] Table 1: NMR data of phenolic acid compounds (EHPA) from Hubei Euphorbia

[0046]

[0047] Its chemical structural formula is shown in formula (I):

[0048]

[0049] Example 2. Preparation of EHPA injection formulation containing phenolic acids from Hubei Province.

[0050] The preparation method of the Hubei Euphorbia pekinensis phenolic acid compound EHPA injection includes the following steps:

[0051] 1) Weigh the raw materials according to the following ratio per 1000mL injection preparation: 0.4g of Hubei Euphorbia pekinensis phenolic acid compound EHPA, 15g of Astragalus polysaccharide, 60g of complex organic solvent, 3g of surfactant, 8g of buffer salt composed of NaH2PO4 and Na2HPO4, 20g of NaCl, 2g of antioxidant, and the balance being water for injection. The complex organic solvent is prepared by mixing ethanol and propylene glycol in a volume ratio of 2:1. The surfactant is composed of Span-80 and Tween-20 in a mass ratio of 2:1. The antioxidant is composed of sodium pyrosulfate and EDTA-2Na in a mass ratio of 2:1.

[0052] 2) Take 30% of the total volume of the injection solution and heat it to 55°C. Then add the weighed Astragalus polysaccharide, the buffer salt composed of NaH2PO4 and Na2HPO4 and NaCl, and stir until it is completely dissolved to obtain solution A.

[0053] 3) Take 50% of the total volume of the injection solution and heat the water for injection to 42°C for later use; heat the weighed compound organic solvent to 42°C, then add the weighed Hubei Euphorbia pekinensis phenolic acid compound EHPA and stir evenly, then add the water for injection that has been heated to 42°C, the weighed surfactant and antioxidant and stir to completely dissolve to obtain solution B.

[0054] 4) Slowly add solution A to solution B, then add the remaining water for injection, stir well, fill and sterilize to obtain Hubei Euphorbia pekinensis phenolic acid compound EHPA injection preparation (tenfold concentration). When using, dilute the injection preparation with physiological saline at a volume ratio of 1:9.

[0055] Example 3: Preparation of EHPA injection formulation containing phenolic acids from Hubei Province

[0056] The preparation method of the Hubei Euphorbia pekinensis phenolic acid compound EHPA injection includes the following steps:

[0057] 1) Weigh the raw materials according to the following ratio per 1000mL injection preparation: 0.2g of Hubei Euphorbia pekinensis phenolic acid compound EHPA, 20g of Astragalus polysaccharide, 50g of complex organic solvent, 2g of surfactant, 8g of buffer salt composed of NaH2PO4 and Na2HPO4, 20g of NaCl, 2g of antioxidant, and the remainder of water for injection. The complex organic solvent is prepared by mixing ethanol and propylene glycol in a volume ratio of 2:1. The surfactant is composed of Span-80 and Tween-20 in a mass ratio of 2:1. The antioxidant is composed of sodium pyrosulfate and EDTA-2Na in a mass ratio of 2:1.

[0058] 2) Take 30% of the total volume of the injection solution and heat it to 55°C. Then add the weighed Astragalus polysaccharide, the buffer salt composed of NaH2PO4 and Na2HPO4 and NaCl, and stir until it is completely dissolved to obtain solution A.

[0059] 3) Take 50% of the total volume of the injection solution and heat the water for injection to 42°C for later use; heat the weighed compound organic solvent to 42°C, then add the weighed Hubei Euphorbia pekinensis phenolic acid compound EHPA and stir evenly, then add the water for injection that has been heated to 42°C, the weighed surfactant and antioxidant and stir to completely dissolve to obtain solution B.

[0060] 4) Slowly add solution A to solution B, then add the remaining water for injection, stir well, fill and sterilize to obtain Hubei Euphorbia pekinensis phenolic acid compound EHPA injection preparation (tenfold concentration). When using, dilute the injection preparation with physiological saline at a volume ratio of 1:9.

[0061] Example 4: Preparation of EHPA injection formulation containing phenolic acids from Hubei Province

[0062] The preparation method of the Hubei Euphorbia pekinensis phenolic acid compound EHPA injection includes the following steps:

[0063] 1) Per 1000 mL of injection preparation, there are 0.3 g of Hubei Euphorbia pekinensis phenolic acid compound EHPA, 15 g of Astragalus polysaccharide, 60 g of complex organic solvent, 3 g of surfactant, 8 g of buffer salt composed of NaH2PO4 and Na2HPO4, 20 g of NaCl and 2 g of antioxidant, wherein the complex organic solvent is prepared by ethanol and propylene glycol in a volume ratio of 2:1, the surfactant is composed of Span-80 and Tween-20 in a mass ratio of 2:1, and the antioxidant is composed of sodium pyrosulfate and EDTA-2Na in a mass ratio of 2:1;

[0064] 2) Take 30% of the total volume of the injection solution and heat it to 55°C. Then add the weighed Astragalus polysaccharide, the buffer salt composed of NaH2PO4 and Na2HPO4 and NaCl, and stir until it is completely dissolved to obtain solution A.

[0065] 3) Take 50% of the total volume of the injection solution and heat the water for injection to 42°C for later use; heat the weighed compound organic solvent to 42°C, then add the weighed Hubei Euphorbia pekinensis phenolic acid compound EHPA and stir evenly, then add the water for injection that has been heated to 42°C, the weighed surfactant and antioxidant and stir to completely dissolve to obtain solution B.

[0066] 4) Slowly add solution A to solution B, then add the remaining water for injection, stir well, fill and sterilize to obtain Hubei Euphorbia pekinensis phenolic acid compound EHPA injection preparation (tenfold concentration). When using, dilute the injection preparation with physiological saline at a volume ratio of 1:9.

[0067] Example 5: Product Stability Test

[0068] The EHPA injection formulations of phenolic acid compounds from Hubei Province prepared in Examples 2-4 were stored at 40°C for accelerated testing. The results of 90 days of observation and testing are shown in Table 2 below:

[0069] Table 2 Product stability test results

[0070]

[0071] Compared to Example 2, Comparative Group 1 did not contain any antioxidants, but its other components and preparation methods were the same as in Example 2. Based on the above accelerated tests, it can be seen that the EHPA injection formulations of euphorbia pekinensis phenolic acid compounds prepared in Examples 2-4 of this invention have strong stability. It is estimated that their shelf life is not less than two years, and the quality and properties of the product are relatively stable within the shelf life.

[0072] Example 6: Safety and Drug Metabolism Tests

[0073] (1) Acute toxicity test

[0074] One hundred and twenty healthy mice (average weight 18.2g) were randomly divided into six groups according to weight and sex. The mice were injected intramuscularly with either 1mL or 2mL of the EHPA injection formulation (diluted 10-fold) from Examples 2-4, respectively. After seven days of observation, no animal deaths or adverse reactions were observed. This indicates that the EHPA injection formulation prepared according to this invention is safe at the normal dosage of 2mL per kilogram of body weight.

[0075] (2) Drug metabolism test

[0076] Ten healthy piglets were accurately weighed, with an average weight of 8.5 kg. Each piglet was injected with 1 mL of the EHPA injection formulation (diluted 10 times) from Example 2 at a rate of 1 mL per kg of body weight. Blood samples were collected at 0.5 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, and 72 h after injection to determine the EHPA content in the blood. The results showed that at 24 h after injection, at least 4.0 μg of EHPA could still be detected per mL of blood. At 48 h, the concentration decreased to 1.0-2.0 μg per mL of blood. At 72 h, a small amount of EHPA was still detectable in the blood. By 96 h, EHPA was undetectable in the blood. This indicates that the EHPA injection formulation prepared in this invention has an efficacy lasting longer than 48 h, a long half-life, and is largely metabolized and excreted within 96 h, demonstrating high safety. Pathological section analysis of the injection sites in necropsy pigs showed no pathological changes, indicating that the injectable preparation was well absorbed, had no adverse reactions, had a long half-life, and could be completely excreted through metabolism, thus demonstrating high safety.

[0077] Example 7: Injection treatment for porcine PEDV infection

[0078] Experimental animals: 15-day-old piglets born to healthy, susceptible pregnant sows (neutralizing antibodies against porcine dyscoronavirus and porcine epidemic diarrhea virus were both no higher than 1:4). Thirty healthy piglets were selected and provided by Shaanxi Nuoweilihua Biotechnology Co., Ltd.

[0079] The virus strain used for challenge was porcine epidemic diarrhea virus strain PEDV-KB2013-4.

[0080] Experimental methods: Piglets were randomly divided into 3 groups, with group 1 being the experimental group. A culture of porcine epidemic diarrhea virus strain PEDV-KB2013-4 (10...) was used. 5.0 TCID 50 Group 1 was challenged with 1 mL of the diluted EHPA injection formulation from Example 2, diluted 10 times, 24 hours later. Treatment was administered every other day at a dose of 0.5 mL (diluted formulation) / kg body weight, for a total of 4 treatments. Group 2 was the control group, treated with a culture of porcine epidemic diarrhea virus strain PEDV-KB2013-4 (10 mL / kg body weight). 5.0 TCID 50Group 1 was challenged with 1 mL of the virus per piglet, with no subsequent drug treatment. Group 3 served as the blank control group, receiving neither challenge nor drug treatment. Animals in Groups 1-3 were isolated and their disease progression and survival were observed for 14 days. Results were as follows: In Group 1, 12 hours after challenge, some piglets exhibited varying degrees of vomiting, decreased appetite, and elevated body temperature. Drug administration was initiated 24 hours after challenge. At 36 hours, some piglets recovered, while a few remained lethargic and prone to lying down. At 72 hours, one piglet died, and the remaining piglets showed minimal symptoms. At the end of the experiment, 9 piglets survived, representing a survival rate of 90%. Group 2 was the challenge group. 12 hours after challenge, some piglets exhibited varying degrees of vomiting, decreased appetite, and elevated body temperature. 24 hours after challenge, one piglet developed watery diarrhea and died, and other piglets also showed diarrhea and vomiting symptoms. 48 hours after challenge, the total number of dead piglets reached 4, and the surviving piglets showed lethargy and swelling and redness around the anus. 72 hours after challenge, the total number of dead piglets reached 8. 120 hours after challenge, all piglets died, resulting in a 100% mortality rate. Group 3 was the blank control group. Piglets in this group showed normal feed intake and weight gain, normal body temperature, and a 100% survival rate at the end of the experiment. Based on the above experiments, it can be concluded that for PEDV-KB2013-4 strain challenge, the EHPA injection preparation has a good effect on controlling diarrhea and can effectively reduce mortality in infected animals, demonstrating excellent therapeutic efficacy.

[0081] Example 8: Clinical efficacy trial

[0082] Two hundred pigs (piglet and fattening pigs) with naturally occurring diarrhea were selected. Testing revealed that all pigs were infected with PEDV, TGEV, or other bacteria. They were randomly divided into two groups according to body weight. Group 1 was treated with commercially available levofloxacin hydrochloride injection (0.2 mL / kg body weight) via intramuscular injection. Group 2 was treated with the undiluted EHPA injection formulation from Example 2, at a dose of 0.5 mL / 10 kg body weight, for three consecutive treatments. Results showed that the EHPA injection formulation achieved a 90% cure rate after one injection, a 95% cure rate after two injections, and a 97% cure rate after three injections. In contrast, the levofloxacin hydrochloride injection formulation achieved a 69% cure rate after one injection, a 75% cure rate after two injections, and an 81% cure rate after three injections. Therefore, the EHPA injection formulation of this invention has superior clinical efficacy compared to levofloxacin hydrochloride injection.

Claims

1. An EHPA injection formulation for treating porcine viral diarrhea, characterized by: 0.2-0.4g EHPA, 10-20g Astragalus polysaccharide, 50-70g compound organic solvent, 2-4g surfactant, 5-10g buffer salt composed of NaH2PO4 and Na2HPO4, 20g NaCl and 1-2g antioxidant per 1000mL injection preparation; The chemical structural formula of the EHPA is shown as formula (I):

2. The EHPA injection preparation for treating porcine viral diarrhea according to claim 1, characterized by, The preparation method of the EHPA is as follows: (1) Ethanol extraction: the naturally air-dried roots of Euphorbia humifusa are crushed by a crusher, and the crushed powder is extracted by ethanol, filtered, and the filtrate is concentrated under reduced pressure to obtain ethanol extract; (2) n-butanol extraction: the extract obtained in step (1) is suspended in water, and then extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the n-butanol extract is concentrated under reduced pressure to obtain n-butanol extract; (3) primary column chromatography: the n-butanol extract obtained in step (2) is subjected to column chromatography separation, the filler is silica gel with a particle size of 200-300 mesh, the eluent is chloroform-methanol with a volume ratio of 15 / 1-1 / 4, gradient elution is performed, the eluent containing the target compound is collected, and the solvent is removed by evaporation under reduced pressure to obtain the primary column chromatography product; (4) secondary column chromatography: the primary column chromatography product obtained in step (3) is subjected to column chromatography separation, the filler is silica gel with a particle size of 200-300 mesh, the eluent is chloroform-methanol with a volume ratio of 100:1-1:1, gradient elution is performed, the eluent containing the target compound is collected, and the solvent is removed by evaporation under reduced pressure to obtain the secondary column chromatography product; (5) preparation and purification: the secondary column chromatography product obtained in step (4) is further separated by gel column LH-20, and then purified by semi-preparative HPLC to obtain the compound shown as formula (I).

3. The EHPA injection preparation for treating porcine viral diarrhea according to claim 2, characterized by, The specific operation method of step (1) is as follows: the crushed root powder of Euphorbia humifusa after drying is extracted by ethanol, the mass-volume ratio of the root powder and ethanol is 1 / 5-1 / 10 (kg / L), the extraction is performed at room temperature for 5-10 days, then filtered to obtain ethanol extract, the filter residue is repeatedly extracted for 2-4 times, the ethanol extracts obtained in each time are combined, the solvent is recovered by evaporation under reduced pressure to obtain ethanol extract; the concentration of the extraction solvent ethanol used in step (1) is 95%, the extraction of n-butanol in step (2) is performed for 2-4 times, preferably 3 times, the n-butanol extraction phases are combined, the solvent is recovered by evaporation under reduced pressure to obtain n-butanol extract, preferably, the eluent of gel column LH-20 in step (5) is 50% methanol; preferably, the mobile phase of semi-preparative HPLC in step (5) is 30% methanol.

4. The EHPA injection formulation for treating porcine viral diarrhea according to claim 1, characterized by, The EHPA injection preparation contains 0.4g EHPA, 15g Astragalus polysaccharide, 60g compound organic solvent, 3g surfactant, 8g buffer salt composed of NaH2PO4 and Na2HPO4, 20g NaCl and 2g antioxidant per 1000mL injection preparation.

5. The EHPA injection preparation for treating porcine viral diarrhea according to claim 1 or 4, characterized by, The complex organic solvent is composed of propylene glycol, dimethylformamide or ethanol, and further preferably, the complex organic solvent is composed of ethanol and propylene glycol in a volume ratio of 2:

1.

6. The EHPA injection formulation for treating porcine viral diarrhea according to claim 1, characterized by, Preferably, the surfactant is selected from one or both of span-80 and Tween-20, and more preferably, the surfactant is composed of span-80 and Tween-20 in a mass ratio of 2:1; the antioxidant is selected from sodium bisulfite, sodium pyrosulfite and / or disodium EDTA, and preferably, the antioxidant is composed of sodium pyrosulfite and disodium EDTA in a mass ratio of 2:

1.

7. The EHPA injection formulation for treating porcine viral diarrhea according to claim 1, characterized by, The EHPA injection preparation is composed of 0.4 g of EHPA, 15 g of astragalus polysaccharide, 60 g of complex organic solvent, 3 g of surfactant, 8 g of buffer salt composed of NaH2PO4 and Na2HPO4, 20 g of NaCl and 2 g of antioxidant per 1000 mL of injection preparation, the complex organic solvent is composed of ethanol and propylene glycol in a volume ratio of 2:1, the surfactant is composed of span-80 and Tween-20 in a mass ratio of 2:1, and the antioxidant is composed of sodium pyrosulfite and disodium EDTA in a mass ratio of 2:

1.

8. The method of claim 1-7 for the preparation of EHPA injection formulation for the treatment of porcine viral diarrhea characterized in that, The method comprises the following steps: 1) weighing the raw materials according to the proportion of 0.2-0.4 g of EHPA, 10-20 g of astragalus polysaccharide, 50-70 g of complex organic solvent, 2-4 g of surfactant, 5-10 g of buffer salt composed of NaH2PO4 and Na2HPO4, 20 g of NaCl, 1-2 g of antioxidant and the rest of injection water per 1000 mL of injection preparation; 2) heating 30% of the total amount of injection water to 55-60℃, then adding the weighed astragalus polysaccharide, buffer salt composed of NaH2PO4 and Na2HPO4 and NaCl, stirring to completely dissolve, to obtain A liquid; 3) heating 50% of the total amount of injection water to 40-45℃ for standby; heating the weighed complex organic solvent to 40-45℃, then adding the weighed EHPA, stirring uniformly, then adding the injection water heated to 40-45℃, the weighed surfactant and antioxidant, stirring to completely dissolve, to obtain B liquid; 4) slowly adding A liquid to B liquid, then adding the rest of the injection water, stirring uniformly, then filling, sterilizing, to obtain the EHPA injection preparation.

Images