Composition for preventing and treating animal hepatitis E and application thereof

Abstract

The present invention belongs to the technical field of veterinary drugs, and specifically relates to a composition and application for preventing and treating animal hepatitis E. The effective ingredients of the composition include lilac leaf extract and liver hydrolyzed peptides. The composition is prepared by mixing lilac leaf extract and liver hydrolyzed peptide extract in a volume ratio of (1-9):3, wherein the tyrosol content in the lilac leaf extract is 1.0-2.5 mg / mL, and the polypeptide content in the liver hydrolyzed peptide extract is 10.0-20.0 mg / mL. The present invention is a composition of pure Chinese medicine and biochemical preparations, which is safe, non-toxic and non-side effects, can be used as drinking water and has no palatability problems. It has a definite therapeutic effect on animal hepatitis E, is green and safe, and is easy to use, and has good market application and promotion value.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drugs, and particularly relates to a composition for preventing and treating animal hepatitis E and an application thereof. Background Art

[0002] Avian Hepatitis E virus (aHEV) is the main pathogen of big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HS) in chickens. aHEV spreads horizontally quickly in chickens, with a high infection rate. It is one of the important diseases that endanger the development of laying hen and breeder chicken farming. The virus mostly infects broiler breeders and laying hens aged 30 to 72 weeks, causing hepatosplenomegaly and liver rupture and bleeding. Poultry infected with HEV will cause the egg production rate of 30 to 72-week-old breeders and laying hens to drop by 20% to 40%, the mortality rate to increase by 1% to 4%, and some chickens will have abdominal congestion and ovarian degeneration, causing huge economic losses to the poultry industry.

[0003] Since 1987, the in vitro cell culture system for hepatitis E virus (HEV) has been continuously developed, and a large number of research results have been obtained on this basis, which has greatly promoted human understanding of the virus. At present, many different cell culture systems have been tested using various HEV strains, but viral replication usually progresses very slowly, and the extremely low number of viral particles makes it impossible to achieve productive replication of HEV after infection. Therefore, vaccine research has been hindered by many obstacles, and there is currently no commercial vaccine. Due to the lack of effective preventive measures and therapeutic drugs to prevent and control the spread of the pathogen, the post-onset treatment measures are first to promptly clean up the source of infection, strengthen feeding management, avoid further infection of healthy chickens, and reduce economic losses; the second is to reduce stress and avoid a decrease in the immunity of the flock. In the current poultry veterinary clinical practice, the harm of avian HEV infection to my country's poultry industry is still seriously underestimated, and the research on related comprehensive prevention and control technologies is also lagging behind.

[0004] Studies have confirmed that liver damage in viral hepatitis is not caused by the direct cytopathic effect of the virus, but by the host's immune response to viral proteins expressed by infected liver cells. In the clinical treatment of human liver diseases, some drugs for the prevention and treatment of human hepatitis E provide some inspiration for the prevention and treatment of avian hepatitis E.

[0005] First of all, Chinese medicine has the characteristics of regulating the body's immunity while clearing away heat and detoxifying, and antibacterial and anti-inflammatory. It is urgent to screen and develop Chinese medicine for the treatment of avian hepatitis E by using my country's unique Chinese medicine theory and rich botanical resources. Lilac leaves are bitter and cold in nature. They enter the stomach, liver, and gallbladder meridians and have the effects of clearing heat and detoxifying, promoting dampness and relieving jaundice. Lilac is reported to have a liver-protecting effect. The "Xinhua Compendium of Materia Medica" records that lilac is mainly used to treat "acute icteric hepatitis, external antibacterial, explosive eye inflammation, and various sores and swellings." In the folk, it is very common to use lilac leaf decoction to treat explosive eye inflammation and prevent and treat dysentery. The "Changbai Mountain Botanicals" records that lilac "treats diarrhea and hepatitis." Modern research shows that lilac leaves have good antiviral, antibacterial and anti-inflammatory, liver-protecting and gallbladder-promoting, antioxidant, enhancing the body's humoral immunity, cooling and lowering blood pressure, and antitussive and expectorant effects, and have relatively small toxic and side effects. Its active ingredients are protocatechuic aldehyde and protocatechuic acid. Capsules and tablets made from lilac have a good effect on the treatment of acute icteric hepatitis and dysentery, and have been included in the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China" for Chinese medicine formula preparations. The main active substance basis of lilac leaves is tyrosol, 3,4-dihydroxyphenylethanol, 3,4-dihydroxybenzoic acid, syringoside, etc. The water extract of lilac leaves has different degrees of inhibition on 22 common bacteria.

[0006] Secondly, animal-derived bioactive peptides can effectively repair liver cell damage and are widely used in clinical practice for the treatment of human liver diseases, but their application in the clinical application of avian hepatitis E is still blank. Liver hydrolyzed peptides are extracted from the liver of healthy cattle by enzymatic hydrolysis and contain peptides, amino acids, and nucleic acid substances. Liver hydrolyzed peptides can promote protein synthesis, reduce protein decomposition, promote the proliferation and regeneration of normal liver cells, have a good protective effect on liver cell damage caused by various reasons, can reduce alanine aminotransferase, and promote the recovery of liver diseased tissue. Liver hydrolyzed peptides are often used in clinical treatment of chronic hepatitis, cirrhosis and other diseases.

[0007] Therefore, the market urgently needs to develop a combination solution for the prevention and treatment of avian hepatitis E that can utilize both the causal therapeutic effects of traditional Chinese medicine and the symptomatic therapeutic effects of bioactive peptides. Summary of the invention

[0008] In view of the problems and shortcomings in the prior art, the present invention aims to provide a composition and application for preventing and treating hepatitis E in animals.

[0009] Based on the above purpose, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a composition for preventing and treating hepatitis E in animals, wherein the effective ingredients of the composition include lilac leaf extract and liver hydrolyzed peptides.

[0011] Preferably, the composition is formed by mixing a lilac leaf extract and a liver hydrolyzed peptide extract in a volume ratio of (1-9):3, wherein the tyrosol content in the lilac leaf extract is 1.0-2.5 mg / mL, and the polypeptide content in the liver hydrolyzed peptide extract is 10.0-20.0 mg / mL.

[0012] Preferably, the preparation method of the lilac leaf extract is: crush the lilac leaves, add water to extract, add cellulase and pectinase to perform enzymatic hydrolysis after the extraction, and heat, filter and reduce pressure to concentrate to obtain the lilac leaf extract after the enzymatic hydrolysis.

[0013] Preferably, the preparation method of the liver hydrolyzed peptide extract is: adding water to the liver to extract to obtain a liver homogenate, adding protease to the liver homogenate for digestion, and heating and filtering after the digestion to obtain the liver hydrolyzed peptide extract.

[0014] Preferably, the extraction is a flash extraction.

[0015] Preferably, the animal is poultry.

[0016] More preferably, the poultry is chicken.

[0017] In a second aspect, the present invention provides a method for preparing a composition for preventing and treating hepatitis E in animals. The composition for preventing and treating hepatitis E in animals is obtained by uniformly mixing a lilac leaf extract and a liver hydrolyzed peptide extract in a volume ratio of (1 to 9):3.

[0018] Preferably, the method for preparing the composition for preventing and treating hepatitis E in animals specifically comprises the following steps:

[0019] (1) grinding lilac leaves and then adding water to perform extraction, adding cellulase and pectinase to perform enzymolysis after the extraction, heating, filtering, and concentrating under reduced pressure to obtain a lilac leaf extract after the enzymolysis;

[0020] (2) adding water to the liver to extract and obtain a liver homogenate, adding protease to the liver homogenate for digestion, and heating and filtering after the digestion to obtain a liver hydrolyzed peptide extract;

[0021] (3) The lilac leaf extract and the liver hydrolyzed peptide extract are mixed evenly to obtain a composition for preventing and treating hepatitis E in animals.

[0022] Preferably, the mesh size of the powder subjected to the pulverization treatment in step (1) is 20 to 40 meshes.

[0023] Preferably, the mass ratio of the lilac leaves to water in step (1) is 1:(8-10).

[0024] Preferably, the extraction in step (1) is a flash extraction, the time of the flash extraction is 5 to 10 minutes, the voltage is 130 to 220 V, and the rotation speed is 8000 to 12000 r / min.

[0025] Preferably, the enzymatic hydrolysis treatment temperature in step (1) is 45-50° C., the pH value is 4.0-4.5, and the time is 3 hours.

[0026] More preferably, the amount of cellulase used is 100,000 to 200,000 IU of cellulase added to 1 kg of lilac leaves, and the amount of pectinase used is 300,000 to 500,000 IU of pectinase added to 1 kg of lilac leaves.

[0027] Preferably, the heating temperature in step (1) is 80-90° C. and the heating time is 0.5-1 h.

[0028] Preferably, the specific process of the filtration in step (1) is: first filter and collect filtrate 1, and boil the filter residue with water, wherein the mass ratio of the filter residue to water is 1:8, and then filter again after boiling, collect filtrate 2, and mix filtrate 1 and filtrate 2.

[0029] More preferably, the decoction time is 1 to 2 hours.

[0030] Preferably, in step (1), the reduced pressure concentration is performed until the ratio of lilac leaf extract to lilac leaf is 1 L:1 kg.

[0031] Preferably, the tyrosol content in the lilac leaf extract is 1.0-2.5 mg / mL.

[0032] Preferably, in step (2), the mass ratio of the liver to water is 1:(1-2).

[0033] More preferably, the liver is bovine liver.

[0034] Preferably, the extraction in step (2) is a flash extraction, the extraction time of the flash extraction is 5 to 10 minutes, the voltage is 130 to 220 V, and the rotation speed is 5000 to 10000 r / min.

[0035] Preferably, the digestion treatment in step (2) is carried out at a temperature of 45 to 50° C., a pH value of 2.5 to 3.5, and a time of 4 to 6 hours.

[0036] More preferably, the amount of the protease used is 500,000 to 600,000 IU of protease added to 1 kg of liver homogenate.

[0037] More preferably, the protease is an acidic protease.

[0038] Preferably, the heating temperature in step (2) is 80-90° C. and the heating time is 15-30 min.

[0039] Preferably, the specific process of the filtration in step (2) is: firstly perform microfiltration treatment using a 0.1 μm filter element, and then perform ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 8k to 10kD.

[0040] Preferably, the hydrolyzed peptide content in the liver hydrolyzed peptide extract in step (2) is 10.0-20.0 mg / mL.

[0041] Preferably, the lilac leaf extract and the liver hydrolyzed peptide extract in step (3) are mixed in a volume ratio of (1-9):3.

[0042] In a third aspect, the present invention provides a drug for preventing and treating animal hepatitis E, the main raw materials of which include the composition for preventing and treating animal hepatitis E described in the first aspect and pharmaceutically acceptable excipients.

[0043] Preferably, the drug is in oral dosage form.

[0044] More preferably, the oral dosage form is any one of oral liquid, granules, powder, extract or soluble powder.

[0045] More preferably, the method for preparing the oral liquid is: sterilizing the composition described in the first aspect to obtain the composition for preventing and treating hepatitis E in animals.

[0046] Preferably, the sterilization is high-pressure steam sterilization, the temperature of the high-pressure steam sterilization is 105-121° C., and the time is 30-40 minutes.

[0047] In a fourth aspect, the present invention provides use of the composition described in the first aspect in the preparation of a drug or pharmaceutical preparation for preventing and treating hepatitis E in animals.

[0048] In a fifth aspect, the present invention provides use of the drug described in the third aspect in the preparation of a pharmaceutical preparation for preventing and treating hepatitis E in animals.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. The present invention is the first to use a combination of lilac leaf extract and liver hydrolyzed peptides to prevent and treat animal hepatitis E. Lilac leaves can clear away heat and detoxify, reduce inflammation, and protect the liver, and have a good preventive and therapeutic effect on animal hepatitis E. At the same time, liver hydrolyzed peptides can promote the proliferation and regeneration of poultry liver cells, accelerate the repair of liver diseased tissues, participate in the life activities of the body, and play a biologically active role in regulating the digestive system, nervous system, endocrine system, and immune function of the animal body. The combination of the two can not only play the causal therapeutic effect of lilac leaves in antiviral and anti-inflammatory, but also play the symptomatic therapeutic effect of liver hydrolyzed peptides in repairing liver cell damage.

[0051] 2. The present invention is the first to explore the compatibility ratio of lilac leaf extract and liver hydrolyzed peptides in combination. In the medication group, lilac leaf extract and liver hydrolyzed peptides were used alone, and the effect of controlling the weight change caused by hepatitis E virus infection in animals was not ideal. After the two were used in combination, the therapeutic effect was significantly improved. Among them, when the volume ratio of lilac leaf extract to liver hydrolyzed peptide extract was 2:1, the effect was the best, and there was no significant difference with the uninfected and unmedicated group (P>0.05).

[0052] 3. The lilac leaf extract and liver hydrolyzed peptide of the present invention use flash extraction coupled with enzyme extraction. Flash extraction can mix the solvent and the solute at a high speed, while the enzymatic extraction can decompose the cellulose and hemicellulose in the lilac leaves and the macromolecular protein in the liver. The coupling of the two methods can fully enrich the active ingredients in the lilac leaves and the liver. Compared with the traditional extraction method, the content of the effective ingredients in the extract is significantly improved.

[0053] 3. The present invention is a composition of pure Chinese medicine and biochemical preparations, which is safe, has no toxic side effects, can be used with drinking water and has no palatability problems. It has a definite effect on animal hepatitis E, is green and safe, and is easy to use, and has good market application and promotion value. DETAILED DESCRIPTION

[0054] The following examples are only for further explanation of the present invention. It should be noted that all technical and scientific terms used in the present invention have the same meanings as in the technical field to which the present invention belongs unless otherwise specified. The experimental methods in the following examples without specifying specific conditions are all based on conventional techniques in the technical field, or according to the conditions recommended by the manufacturer; the reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0055] In order to make the purpose, technical solution and advantages of the present invention clearer and more specific, the present invention is further described in detail by examples below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] Example 1

[0057] A composition for preventing and treating animal hepatitis E, wherein the composition is prepared by mixing a lilac leaf extract and a liver hydrolyzed peptide extract in a volume ratio of 2:1, wherein the lilac leaf extract has a tyrosol content of 2.1 mg / mL, and the liver hydrolyzed peptide extract has a polypeptide content of 15.0 mg / mL.

[0058] The steps of the preparation method of the above composition are as follows:

[0059] (1) Crush lilac leaves to 20 meshes, add purified water for flash extraction, the mass ratio of lilac leaves to purified water is 1:10, the flash extraction time is 8 minutes, the voltage is 200V, the rotation speed is 10000r / min, after the extraction is completed, the temperature is adjusted to 50°C, the pH value is 4.5, and cellulase and pectinase are added for enzymatic hydrolysis for 3 hours, the amount of cellulase used is 200,000 IU of cellulase added to 1 kg of lilac leaves, and the amount of pectinase used is 500,000 IU of cellulase added to 1 kg of lilac leaves. 100,000 IU pectinase, after the enzymatic hydrolysis is completed, heat at 90°C for 0.5h, then filter and collect filtrate 1, and add purified water to the filter residue and boil for 1h, the mass ratio of the filter residue to purified water is 1:8, and filter and collect filtrate 2 after boiling, mix filtrate 1 and filtrate 2 to obtain lilac leaf filtrate, and reduce the pressure and concentrate the lilac leaf filtrate to obtain lilac leaf extract, the reduced pressure and concentration is to the ratio of lilac leaf extract to lilac leaf of 1L:1kg, and the tyrosol content in the lilac leaf extract is 2.1mg / mL;

[0060] (2) adding purified water to the bovine liver for flash extraction, wherein the mass ratio of the bovine liver to the purified water is 1:2, the flash extraction time is 7 minutes, the voltage is 200 V, and the rotation speed is 8000 r / min to obtain a bovine liver homogenate, adjusting the temperature of the bovine liver homogenate to 50° C. and the pH value to 3.5, adding acidic protease for digestion treatment for 5 hours, and the amount of acidic protease used is 600,000 IU of protease added to 1 kg of the bovine liver homogenate. After the digestion treatment is completed, heating is performed at 85° C. for 25 minutes, and then cooling to room temperature, centrifuging to obtain a supernatant, and the supernatant is first microfiltered with a 0.1 μm filter element, and then ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 8 kD to obtain a liver hydrolyzed peptide extract, wherein the hydrolyzed peptide content in the liver hydrolyzed peptide extract is 15.0 mg / mL;

[0061] (3) The lilac leaf extract and the liver hydrolyzed peptide extract are mixed evenly in a volume ratio of 2:1 to obtain a composition for preventing and treating hepatitis E in animals.

[0062] (I) Study on the ratio of drug components

[0063] Example 2

[0064] A medicine for preventing and treating animal hepatitis E, which is prepared by sterilizing the composition for preventing and treating animal hepatitis E prepared in Example 1 with high-pressure steam at 121° C. for 40 minutes. The medicine is an oral liquid.

[0065] Example 3

[0066] A drug for preventing and treating animal hepatitis E, which is basically the same as that of Example 2, except that a different ratio of the composition for preventing and treating animal hepatitis E is used. In the composition for preventing and treating animal hepatitis E, a lilac leaf extract and a liver hydrolyzed peptide extract are mixed in a volume ratio of 1:1.

[0067] The preparation method of the above medicine is the same as that of Example 2.

[0068] Example 4

[0069] A drug for preventing and treating animal hepatitis E, which is basically the same as that of Example 2, except that a different ratio of the composition for preventing and treating animal hepatitis E is used. In the composition for preventing and treating animal hepatitis E, a lilac leaf extract and a liver hydrolyzed peptide extract are mixed in a volume ratio of 3:1.

[0070] The preparation method of the above medicine is the same as that of Example 2.

[0071] Example 5

[0072] A drug for preventing and treating animal hepatitis E, which is basically the same as that of Example 2, except that a different ratio of the composition for preventing and treating animal hepatitis E is used. In the composition for preventing and treating animal hepatitis E, a lilac leaf extract and a liver hydrolyzed peptide extract are mixed in a volume ratio of 1:2.

[0073] The preparation method of the above medicine is the same as that of Example 2.

[0074] Example 6

[0075] A drug for preventing and treating animal hepatitis E, which is basically the same as that of Example 2, except that a different ratio of the composition for preventing and treating animal hepatitis E is used. In the composition for preventing and treating animal hepatitis E, a lilac leaf extract and a liver hydrolyzed peptide extract are mixed in a volume ratio of 1:3.

[0076] The preparation method of the above medicine is the same as that of Example 2.

[0077] Comparative Example 1

[0078] A medicine for preventing and treating animal hepatitis E, the medicine being a lilac leaf extract; the tyrosol content in the lilac leaf extract is 2.1 mg / mL.

[0079] The above-mentioned drug preparation method comprises the following steps:

[0080] (1) Crush lilac leaves to 20 meshes, add purified water for flash extraction, the mass ratio of lilac leaves to purified water is 1:10, the flash extraction time is 8 minutes, the voltage is 200V, the speed is 10000r / min, after the extraction is completed, the temperature is adjusted to 50°C, the pH value is 4.5, and cellulase and pectinase are added for enzymatic hydrolysis for 3 hours, the amount of cellulase is 200,000 IU of cellulase in 1 kg of lilac leaves, and the amount of pectinase is 500,000 IU of cellulase in 1 kg of lilac leaves. 10,000 IU pectinase, heat at 90°C for 0.5h after the enzymolysis treatment, then filter and collect filtrate 1, add purified water to the filter residue and boil for 1h, the mass ratio of the filter residue to purified water is 1:8, filter and collect filtrate 2 after boiling, mix filtrate 1 and filtrate 2 to obtain lilac leaf filtrate, and reduce the pressure and concentrate the lilac leaf filtrate to obtain lilac leaf extract, the reduced pressure and concentration is to the ratio of lilac leaf extract to lilac leaf is 1L:1kg, and the content of tyrosol in the lilac leaf extract is 2.1mg / mL;

[0081] (2) The lilac leaf extract is sterilized by high pressure steam at 121° C. for 40 minutes to obtain a drug for preventing and treating hepatitis E in animals.

[0082] Comparative Example 2

[0083] A medicine for preventing and treating animal hepatitis E, the medicine is a liver hydrolyzed peptide extract; the content of hydrolyzed peptides in the liver hydrolyzed peptide extract is 15.0 mg / mL.

[0084] The above-mentioned drug preparation method comprises the following steps:

[0085] (1) adding purified water to the bovine liver for flash extraction, wherein the mass ratio of the bovine liver to the purified water is 1:2, the flash extraction time is 7 minutes, the voltage is 200 V, and the rotation speed is 8000 r / min, to obtain a bovine liver homogenate, adjusting the temperature of the bovine liver homogenate to 50° C. and the pH value to 3.5, adding acidic protease for digestion treatment for 5 hours, the amount of acidic protease used is 600,000 IU of protease added to 1 kg of the bovine liver homogenate, heating at 85° C. for 25 minutes after the digestion treatment, then cooling to room temperature, centrifuging to obtain a supernatant, first microfiltering the supernatant with a 0.1 μm filter element, and then ultrafiltering with an ultrafiltration membrane with a molecular weight cutoff of 8 kD to obtain a liver hydrolyzed peptide extract, wherein the hydrolyzed peptide content in the liver hydrolyzed peptide extract is 15.0 mg / mL;

[0086] (2) The liver hydrolyzed peptide extract is sterilized by high pressure steam at 121° C. for 40 minutes to obtain a drug for preventing and treating animal hepatitis E.

[0087] In order to verify the efficacy of drugs for preventing and treating animal hepatitis E on aHEV-infected chickens, the following in vivo pharmacodynamics test was performed.

[0088] Test strain: YT-aHEV strain (GenBank No: MZ736614) was amplified and preserved in the Preventive Veterinary Laboratory of Nanjing Agricultural University.

[0089] Test Example 1:

[0090] 1. Experimental subjects and groups

[0091] 135 specific pathogen-free (SPF) eggs (purchased from Jinan Spafas Poultry Co., Ltd.) were randomly divided into 9 groups A to I, each group of 15 eggs, and the normal development to 14-day-old chicken embryos, the tested strains were intravenously inoculated into 14-day-old SPF chicken embryos of groups A to H, the inoculation dose was 150TCID50, the specific method was as follows: take the SPF chicken embryo, after looking at it on the egg inspection lamp, find a clear vein and use a pencil to frame a length of 1cm, which is the edge of opening the eggshell. Cut the eggshell with a saw blade along the marked pencil edge to ensure that the shell membrane is not damaged. Use a 1mL syringe to draw 0.2ml of the virus liquid to be inoculated to pierce the shell membrane and insert the needle along the vein, then slowly inject the virus liquid into the chicken embryo vein, finally seal the hole with tape and paraffin melting, and continue to incubate in an incubator.

[0092] 2. Experimental Plan

[0093] The chickens in groups A to I were given medication 1 day after hatching, with a dosage of 1 ml / chicken / day. The dosage was as follows: 1 ml of the drug was dissolved in 1 L of water, and the drug was consumed within 4 hours. The drug was used for 7 consecutive days. Group H was the infection group without medication, Group I was the non-infection group without medication, and the rest were infection groups with medication. The specific experimental scheme is shown in Table 1.

[0094] Table 1 Experimental scheme of Test Example 1

[0095]

[0096] 3. Experimental indicators

[0097] 3.1 Weigh all experimental animals 1 day, 1 week, 2 weeks, and 3 weeks after hatching to evaluate the effect of viral infection on the growth of the flock, and also evaluate the effect of medication after infection on the growth of the flock.

[0098] 3.2 Blood samples were collected at 1 week, 2 weeks, and 3 weeks after hatching, and serum was separated from them to detect the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

[0099] 4. Experimental Results

[0100] 4.1 Average weight experimental results

[0101] The average body weight of the experimental animals in each group 1 day (d), 1 week (w), 2 weeks (w), and 3 weeks (w) after infection is shown in Table 2.

[0102] Table 2 Average body weight of experimental animals in each group 1d, 1w, 2w and 3w after infection

[0103] Group 1d 1w 2w 3w Group A 35.11±1.50 43.37±1.21 65.45±1.13 92.93±3.32 Group B 35.12±1.68 43.31±1.02 64.09±1.19 90.81±1.86 Group C 35.07±1.69 43.37±1.25 64.95±1.06 92.34±2.61 Group D 35.10±1.61 43.27±1.09 63.98±1.19 90.68±1.36 Group E 35.13±1.88 43.30±1.20 63.87±1.14 90.61±1.48 Group F 35.10±1.38 43.49±1.06 64.61±1.06 90.59±2.02 Group G 35.17±1.80 43.27±1.19 63.75±1.12 89.07±1.92 Group H 35.23±1.62 43.37±0.81 62.81±0.85 88.49±3.19 Group I 35.11±1.45 43.29±1.05 65.49±1.11 93.77±1.15

[0104] Combining the data in Table 2 with SPSS24.0 analysis, it can be seen that there was no significant difference in body weight between the infected group and the uninfected group 1 day after hatching (P>0.05), indicating that the infection of the YT strain did not cause a significant impact on the growth and development of the chicken embryos from 14 days to 21 days of age.

[0105] At 1 week after hatching, there was still no significant difference in body weight between the infected and uninfected groups (P>0.05), and the weight level of the chickens was basically the same. At 2 weeks after hatching, the average body weight of the chickens was different: the difference between group H and group I was extremely significant (P<0.01), the difference between group A and group I was not significant (P>0.05), the difference between group A and group G was extremely significant (P<0.01), the difference between group A and group H was extremely significant (P<0.01), and the difference between group G and group H was not significant (P<0.05).

[0106] At 3 weeks after hatching: there was an extremely significant difference between group H and group I (P<0.01), there was no significant difference between group A and group I (P>0.05), there was an extremely significant difference between group A and group G (P<0.01), there was an extremely significant difference between group A and group H (P<0.01), there was no significant difference between group G and group H (P<0.05).

[0107] The above experimental results show that the weight results of different experimental groups differed from 2 weeks of age, among which the weight difference between the infected and unmedicated group (group H) and the uninfected and unmedicated group (group I) was significant (p < 0.05), indicating that hepatitis E virus has a significant effect on the weight gain of chicks. In the medication group, the lilac leaf extract (group F) and liver hydrolyzed peptide (group G) were used alone, and the effect of controlling the weight change caused by hepatitis E virus infection was not ideal. After the two were used in combination (groups A to E), the therapeutic effect was significantly improved. Among them, the lilac leaf and liver hydrolyzed peptide were 2:1 (group A), and the effect was the best, and there was no significant difference with the uninfected and unmedicated group (group I) (P > 0.05).

[0108] 4.2.1AST activity test results

[0109] The results of AST enzyme activity testing of experimental animals in each group at 1 week (w), 2 weeks (w), and 3 weeks (w) after hatching are shown in Table 3.

[0110] Table 3 AST activity test results of each group 1w, 2w and 3w after hatching

[0111] Group 1w 2w 3w Group A 14.11±0.47 11.23±0.48 10.04±0.36 Group B 18.88±0.53 14.86±0.74 12.90±0.41 Group C 18.84±0.48 14.95±0.29 12.56±0.51 Group D 18.50±0.44 14.86±0.39 12.37±0.34 Group E 18.50±0.52 14.84±0.66 12.90±0.42 Group F 20.91±0.61 18.00±0.59 14.94±0.42 Group G 19.86±0.77 18.85±0.98 15.17±0.47 Group H 23.48±0.87 22.58±1.32 18.07±0.54 Group I 10.53±0.42 10.24±0.48 10.26±0.43

[0112] Combining the data in Table 3 with SPSS24.0 analysis, it can be seen that the AST enzyme activity in the infected group (group H) is always significantly higher than that in the uninfected group (group I) (P<0.01). At 3 weeks after hatching, the AST enzyme activity in group A was close to that in group I (P>0.05), and the other groups were still higher than group I. The AST enzyme activity in groups B, C, D, and E was higher than that in group A with significant differences (P<0.05), and lower than that in group H with significant differences (p<0.05).

[0113] The above experimental results show that when the ratio of lilac leaves to liver hydrolyzed peptides is 2:1 (Group A), it has a significant effect on reducing the activity of AST enzyme after infection. At 3 weeks after hatching, although the infected group (Group H) did not use drugs, the AST enzyme activity level also showed a downward trend, indicating that the disease may heal itself after 3 weeks. The AST enzyme activity of other groups also showed a downward trend, and the AST enzyme activity was lower than that of the infected group (Group H), indicating that the drugs in other groups also had a certain effect on reducing the activity of AST enzyme, and the effect of lilac leaf extract (Group F) and liver hydrolyzed peptides (Group G) alone was not as significant as the effect of the combination of the two (Groups A to E). Among them, the effect of lilac leaves and liver hydrolyzed peptides was the best when the ratio was 2:1 (Group A).

[0114] 4.2.2 ALT activity test results

[0115] The results of ALT enzyme activity detection of experimental animals in each group at 1 week (w), 2 weeks (w), and 3 weeks (w) after hatching are shown in Table 4.

[0116] Table 4 ALT activity test results of each group 1w, 2w and 3w after hatching

[0117] Group 1w 2w 3w Group A 8.64±0.66 6.62±0.43 6.70±0.74 Group B 11.88±0.65 10.70±0.42 9.26±0.46 Group C 11.75±0.51 10.82±0.70 9.23±0.37 Group D 11.69±0.73 10.29±0.82 9.28±0.54 Group E 11.68±0.85 10.55±0.81 9.22±0.53 Group F 14.60±0.46 11.80±0.50 10.05±0.37 Group G 14.88±0.43 12.03±0.53 9.97±0.49 Group H 18.85±0.64 15.85±0.22 13.96±0.34 Group I 5.99±0.70 6.13±0.54 5.79±0.51

[0118] Combining the data in Table 4 with SPSS24.0 analysis, it can be seen that the ALT enzyme activity in the infected group (group H) was always significantly higher than that in the uninfected group (group I) (P < 0.01). At 2 weeks and 3 weeks after hatching, the ALT enzyme activity levels of each infected group decreased. Among them, at 3 weeks after hatching, the AST enzyme activity of group A was close to that of group I (P > 0.05), and the other groups were still higher than group I and the difference was extremely significant (P < 0.01). The ALT enzyme activity of groups B, C, D, and E was higher than that of group A and the difference was significant (P < 0.05), and lower than that of group H and the difference was significant (p < 0.05).

[0119] The above experimental results show that when the ratio of lilac leaves to liver hydrolyzed peptides is 2:1 (Group A), it has a significant effect on reducing the activity of ALT enzyme after infection. At 3 weeks after hatching, although the infected group (Group H) did not use drugs, the ALT enzyme activity level also showed a downward trend, indicating that the disease may heal itself after 3 weeks. The ALT enzyme activity of other groups also showed a downward trend, and the AST enzyme activity was lower than that of the infected group (Group H), indicating that the drugs in other groups also had a certain effect on reducing the activity of ALT enzyme, and the effect of lilac leaf extract (Group F) and liver hydrolyzed peptides (Group G) alone was not as significant as the effect of the combination of the two (Groups A to E). Among them, the effect of lilac leaves and liver hydrolyzed peptides was the best when the ratio was 2:1 (Group A).

[0120] The above experimental results show that the ratio of lilac leaf extract and liver hydrolyzed peptide in Example 2 is optimal and has the best therapeutic effect on hepatitis E.

[0121] (II) Research on extraction methods of drug components

[0122] Comparative Example 3

[0123] A medicine for preventing and treating animal hepatitis E. The composition is prepared by mixing a lilac leaf extract and a liver hydrolyzed peptide extract in a volume ratio of 2:1, wherein the lilac leaf extract has a tyrosol content of 1.63 mg / mL, and the liver hydrolyzed peptide extract has a polypeptide content of 15.0 mg / mL.

[0124] The preparation method of the above-mentioned drug is basically the same as that of Example 2, except that, in step (1), the extraction method of lilac leaf extract is: crushing lilac leaves to 20 meshes, adding 70% ethanol and heating under reflux for extraction for 1.5 hours, the mass ratio of lilac leaves to 70% ethanol is 1:10, filtering and collecting filtrate 1, and adding 70% ethanol to the residue and heating under reflux for extraction for 1.5 hours, the mass ratio of the residue to 70% ethanol is 1:8, after the extraction is completed, filtering and collecting filtrate 2, mixing filtrate 1 and filtrate 2 to obtain lilac leaf filtrate, reducing pressure and concentrating the lilac leaf filtrate, recovering ethanol, and obtaining lilac leaf extract, wherein the reduced pressure and concentration is to a ratio of lilac leaf extract to lilac leaf of 1L:1kg.

[0125] Comparative Example 4

[0126] A medicine for preventing and treating animal hepatitis E. The composition is prepared by mixing a lilac leaf extract and a liver hydrolyzed peptide extract in a volume ratio of 2:1, wherein the lilac leaf extract has a tyrosol content of 1.73 mg / mL and the liver hydrolyzed peptide extract has a polypeptide content of 15.0 mg / mL.

[0127] The preparation method of the above-mentioned drug is basically the same as that of Example 2, except that in step (1), the extraction method of the lilac leaf extract is: crush the lilac leaves to 20 mesh, add purified water and boil for 1 hour, the mass ratio of the lilac leaves to the purified water is 1:10, after boiling, the temperature is adjusted to 50°C, the pH value is 4.5, and cellulase and pectinase are added for enzymatic hydrolysis for 3 hours, the amount of the cellulase is 200,000 IU of cellulase and pectinase is added to 1 kg of lilac leaves. The dosage is to add 500,000 IU pectinase to 1 kg of lilac leaves, heat at 90°C for 0.5 h after the enzymatic hydrolysis, then filter and collect filtrate 1, and add purified water to the residue and boil for 1 h, the mass ratio of the residue to purified water is 1:8, and filter and collect filtrate 2 after boiling, mix filtrate 1 and filtrate 2 to obtain lilac leaf filtrate, and reduce the pressure and concentrate the lilac leaf filtrate to obtain lilac leaf extract, and the reduced pressure and concentration is to a ratio of lilac leaf extract to lilac leaf of 1 L:1 kg.

[0128] The main components of the lilac leaf extract include tyrosol, chlorogenic acid, syringoside, syringin and protocatechuic acid. The effective components of the lilac leaf medicine obtained in Example 2 and Comparative Examples 3 and 4 are respectively measured and are shown in Table 5.

[0129] Table 5 Effects of different extraction methods on the active ingredients of drugs

[0130]

[0131] As can be seen from Table 5, when extracting lilac leaf extract, compared with the traditional alcohol extraction and enzymatic hydrolysis methods of traditional Chinese medicine, the present invention adopts flash extraction and enzyme extraction, and the content of effective ingredients in lilac leaf extract is significantly improved. This is because flash extraction, on the one hand, destroys the cell structure of the plant through high-speed stirring and shearing, and on the other hand, it can mix the solvent and solute at a high speed, improve the contact efficiency of the solvent and solute, and then promote the dissolution of effective ingredients. Enzymatic extraction can decompose cellulose and hemicellulose in lilac leaves, further lyse the cell structure, and release small molecules. The combination of the two methods can fully enrich the active ingredients in lilac leaves.

[0132] In order to verify the efficacy of drugs prepared by different extraction methods on aHEV-infected chickens, the following in vivo pharmacodynamic tests were performed.

[0133] Test Example 2:

[0134] 1. Experimental subjects and groups

[0135] The laying hen farm of Lv in Xiantao, Hubei Province has 30,000 chickens in stock, of which the breed is sea blue brown, the age is 330 days, and the egg production rate is 91%. In May 2023, hepatitis E virus (HEV) infection was found in many commercial laying hen farms around Lv, causing big liver and spleen disease (BLS) in chickens. The breeder farm began to strengthen the biosafety measures of the farm, but the chickens still died of unexplained reasons, with a mortality rate of more than 7%, and the egg production rate dropped by 30% within 10 days. Most of the dead chickens had liver lesions after autopsy, and the liver was obviously enlarged, and even bleeding and necrosis, resulting in heavy losses. The RT-PCR test results of the diseased materials showed that the chickens were infected with hepatitis E virus, and adenovirus and avian leukosis virus were negative. After recommending the invented drug to the customer, the customer had a strong desire to test it. Suspected chickens were selected from the flock, cloacal swabs were collected, and the RT-PCR method was used to test them one by one. 120 positive chickens were selected to participate in the experiment, and they were divided into 4 groups A to D on average, with 30 chickens in each group.

[0136] 2. Experimental Plan

[0137] Chickens in groups A to C started taking the medicine at the same time, with a dosage of 1 ml / day, and the method of use was: dissolve 1 ml of the medicine in 500 ml of water, drink it after 4 hours, and use it for 7 consecutive days. Among them, group A used the medicine prepared in Example 2, group B used the medicine prepared in Comparative Example 3, group C used the medicine prepared in Comparative Example 4, and group D did not take the medicine. The specific experimental scheme is shown in Table 6.

[0138] Table 6 Experimental scheme of Test Example 2

[0139]

[0140] 3. Experimental indicators and data statistics

[0141] The survival number, mortality rate, average total egg production and egg production rate of all experimental animals were counted on the 0th, 3rd, 5th and 10th days after medication, respectively. The calculation method of mortality rate and egg production rate was based on the 3rd day as an example. Mortality rate on the 3rd day = (number of survivors on the 0th day - number of survivors on the 3rd day) / number of survivors on the 0th day × 100%, egg production rate on the 3rd day = average total egg production on the 3rd day / number of survivors on the 3rd day × 100%. The effect of virus infection on the growth of the flock was evaluated, and the effect of medication after infection on the growth of the flock was also evaluated.

[0142] 4. Experimental Results

[0143] The specific conditions of the experimental animals in each group on the 0th, 3rd, 5th and 10th days are shown in Table 7.

[0144] Table 7 Specific conditions of drug administration of experimental animals in each group on day 0, 3, 5 and 10

[0145]

[0146] As can be seen from Table 7, the drug for preventing and treating animal hepatitis E prepared in Example 2 of the present invention has significant efficacy, the mortality rate on the 10th day is reduced to 0, and the egg production rate is increased to 89%, which is close to the egg production rate (91%) of the chickens before the onset of the disease, and the efficacy is better than the compositions extracted in Comparative Examples 3 and 4.

[0147] This shows that the extraction method of the lilac leaf extract and liver hydrolyzed peptide in Example 2 is optimal and has the best therapeutic effect on hepatitis E.

[0148] In summary, the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value. The above embodiments serve to illustrate the substantial content of the present invention, but do not limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A composition for preventing and treating chicken hepatitis E caused by YT-aHEV strain: GenBank No. MZ736614, characterized in that: The composition is prepared by mixing a lilac leaf extract and a liver hydrolyzed peptide extract in a volume ratio of (1-9):3, wherein the lilac leaf extract has a tyrosol content of 1.0-2.5 mg / mL, and the liver hydrolyzed peptide extract has a polypeptide content of 10.0-20.0 mg / mL; The preparation method of the lilac leaf extract comprises: crushing the lilac leaves, adding water to extract, adding cellulase and pectinase to perform enzymolysis after the extraction, heating, filtering, and concentrating under reduced pressure to obtain the lilac leaf extract after the enzymolysis; The preparation method of the liver hydrolyzed peptide extract comprises: adding water to the liver to extract to obtain a liver homogenate, adding protease to the liver homogenate for digestion, and heating and filtering after the digestion to obtain the liver hydrolyzed peptide extract; The extraction in the preparation process of the liver hydrolyzed peptide extract is flash extraction.

2. A drug for preventing and treating chicken hepatitis E caused by YT-aHEV strain: GenBank No. MZ736614, characterized in that: The medicine comprises the composition according to claim 1 and pharmaceutically acceptable excipients.

3. The drug for preventing and treating chicken hepatitis E caused by YT-aHEV strain: GenBank No. MZ736614 as claimed in claim 2, characterized in that: The drug is in oral dosage form.

4. The drug for preventing and treating chicken hepatitis E caused by YT-aHEV strain: GenBank No. MZ736614 as claimed in claim 3, characterized in that: The oral dosage form is any one of oral liquid, granules, powders, extracts or soluble powders.

5. Use of the composition according to claim 1 in the preparation of a drug or a pharmaceutical preparation for preventing and treating chicken hepatitis E caused by YT-aHEV strain: GenBank No. MZ736614.

6. Use of the drug according to any one of claims 2 to 4 in the preparation of a pharmaceutical preparation for preventing and treating chicken hepatitis E caused by YT-aHEV strain: GenBank No. MZ736614.

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