Chinese herbal medicine feed additive for preventing and treating avian influenza and preparation method of Chinese herbal medicine feed additive

Abstract

The invention provides a Chinese herbal medicine feed additive for preventing and treating avian influenza and a preparation method thereof, and belongs to the technical field of traditional Chinese veterinary medicines. The Chinese herbal medicine feed additive for preventing and treating avian influenza is prepared from the Chinese herbal medicines of isaria cicadae miq, iridis tectori, arisaema cum bile, solidago decurrens, radix pseudostellariae, coriolus versicolor and liquorice, is safe and non-toxic, can remarkably inhibit proliferation of H9N2 subtype AIV in chick embryos under in-vitro conditions, and has a remarkable antiviral effect. Meanwhile, the treatment effect on laying hens suffering from the avian influenza is remarkable, the death rate caused by the avian influenza is remarkably reduced, and the avian influenza can be effectively prevented. The Chinese herbal medicine feed additive disclosed by the invention has huge application potential in the aspects of treating and preventing avian influenza.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese veterinary medicine technology, specifically to a traditional Chinese herbal feed additive for the prevention and treatment of avian influenza and its preparation method. Background Technology

[0002] Avian influenza virus (AIV) is a highly contagious pathogen that can cause severe respiratory diseases in poultry and wild birds. In recent years, the large-scale spread of avian influenza virus worldwide has attracted widespread attention, posing a serious threat to poultry farming, public health and safety, and the ecological environment.

[0003] Vaccination is currently the most important and effective means of preventing AIV (Avian influenza). However, the time from vaccination to the production of protective antibodies is relatively long, and there are issues such as the window period for antibody production after immunization. More importantly, due to the high antigenic variability of AIV, even different strains of the same serotype show significant differences. Furthermore, the short protection period of vaccines and the increasing use of vaccines during the breeding season lead to persistent immune pressure and significantly reduced vaccine effectiveness, which often affects the control of AIV. These shortcomings make research on anti-AIV drugs essential, as they can simultaneously play a preventive and therapeutic role, thereby minimizing the threat of AIV. Currently, although some Western antiviral drugs are used for the prevention and treatment of avian influenza, their efficacy is not ideal. Therefore, finding safe and effective drugs against avian influenza viruses is an urgent need. Traditional Chinese medicine, as a unique drug system with a long history and abundant resources, has great research and application potential. Moreover, the application of traditional Chinese medicine in the prevention and treatment of infectious diseases has a history of thousands of years and plays an important role in combating emerging viral diseases. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a traditional Chinese medicine feed additive for the prevention and treatment of avian influenza and its preparation method.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] First, the present invention provides a traditional Chinese medicine feed additive for the prevention and treatment of avian influenza, the traditional Chinese medicine feed additive being made from the following raw materials in parts by weight: 20-40 parts of Cordyceps militaris, 20-40 parts of Arisaema cum Bile, 20-40 parts of Solidago virgaurea, 20-40 parts of Belamcanda chinensis, 20-40 parts of Codonopsis pilosula, 20-40 parts of Trametes versicolor, and 5-20 parts of Glycyrrhiza uralensis.

[0009] Furthermore, the herbal feed additive is made from the following raw materials in parts by weight: 30-40 parts of Cordyceps militaris, 25-35 parts of Arisaema cum Bile, 25-35 parts of Solidago virgaurea, 30-40 parts of Belamcanda chinensis, 20-30 parts of Codonopsis pilosula, 20-30 parts of Ganoderma lucidum, and 10-20 parts of Glycyrrhiza uralensis.

[0010] Furthermore, the herbal feed additive is made from the following ingredients in parts by weight: 36 parts Cordyceps militaris, 30 parts Arisaema cum Bile, 28 parts Solidago virgaurea, 32 parts Belamcanda chinensis, 26 parts Codonopsis pilosula, 24 parts Ganoderma lucidum, and 14 parts Glycyrrhiza uralensis.

[0011] Secondly, the present invention also provides a method for preparing a traditional Chinese medicine feed additive for the prevention and treatment of avian influenza, wherein the traditional Chinese medicine feed additive is prepared according to the following steps:

[0012] (1) Weigh each raw material according to the following weight parts: 20-40 parts of Cordyceps militaris, 20-40 parts of Arisaema cum Bile, 20-40 parts of Solidago virgaurea, 20-40 parts of Belamcanda chinensis, 20-40 parts of Codonopsis pilosula, 20-40 parts of Ganoderma lucidum, and 5-20 parts of Glycyrrhiza uralensis. Wash, dry and then pulverize and mix evenly to obtain the Chinese medicine raw materials.

[0013] (2) Add 10-14 times the amount of purified water to the Chinese herbal raw materials, soak for more than 30 minutes, bring to a boil over high heat and keep it at a simmer for more than 2 hours, then filter and separate the first decoction. Add 8-10 times the amount of purified water to the residue again, bring to a boil over high heat and keep it at a simmer for more than 1.5 hours, then filter and separate the second decoction. Add 6-8 times the amount of purified water to the residue again, bring to a boil over high heat and keep it at a simmer for more than 1 hour, then filter and separate the third decoction. Combine the three decoctions and refrigerate for 12 hours.

[0014] (3) After standing, filter the decoction, discard the precipitated dregs, collect the clear filtrate, and set aside for later use;

[0015] (4) The clarified filtrate was concentrated under reduced pressure at 60-70℃ and -0.08MPa to -0.1MPa until it became a clear extract with a relative density of 1.15-1.25 at 60℃;

[0016] (5) The extract is spray-dried to make a dry extract powder with a moisture content of less than 5% for later use;

[0017] (6) Anhydrous glucose and soluble starch are mixed at a mass ratio of 2:1~1.5 as excipients. Then, the dry extract powder is mixed with the excipients at a mass ratio of 1:2~2.5. After mixing evenly, the mixture is passed through a 120-150 mesh sieve to obtain the soluble powder of the Chinese herbal feed additive.

[0018] This invention relates to a herbal feed additive made from the following Chinese medicinal herbs: Cordyceps militaris, Arisaema cum Bile, Solidago virgaurea, Belamcanda chinensis, Pseudostellaria heterophylla, Trametes versicolor, and Glycyrrhiza uralensis. The relevant descriptions of each of these herbs are as follows:

[0019] Cordyceps flower: a medicinal fungus of the Cordyceps family, formed by cicada nymphs infected with Penicillium cicadae, also known as "Cicada Flower"; it is sweet in taste and cold in nature, and enters the lung, liver and kidney meridians; it has the effects of dispersing wind-heat, promoting rash eruption, calming wind and stopping spasms, and improving eyesight and clearing away corneal opacity; it is used for symptoms such as exogenous wind-heat, fever, dizziness, red and swollen eyes.

[0020] Arisaema cum Bile: This is made by processing fine powder of Arisaema heterophyllum with bile from cattle, sheep, or pigs, or by fermenting fine powder of raw Arisaema heterophyllum with bile from cattle, sheep, or pigs. It has the effects of clearing heat and resolving phlegm, calming wind and relieving convulsions. It is mainly used for cough with phlegm and heat, thick yellow phlegm, stroke with phlegm-induced coma, and epilepsy.

[0021] Goldenrod: It is the dried whole herb of Solidago decurrens, a plant in the Asteraceae family. It is pungent, bitter, and cool in nature, and enters the lung and liver meridians. It has the effects of clearing heat and detoxifying, reducing swelling and relieving pain. It is often used for symptoms such as wind-heat cold, sore throat, and snake bites.

[0022] Iris tectorum Maxim., a plant of the Iridaceae family, is a dried rhizome. It has the effects of clearing heat and detoxifying, resolving phlegm, and relieving sore throat. It is mainly used to treat heat-toxin phlegm-fire stagnation, sore throat, excessive phlegm, cough and asthma.

[0023] Codonopsis pilosula: The dried tuberous root of Codonopsis pilosula, a plant of the Caryophyllaceae family. It is neutral in nature, sweet and slightly bitter in taste, and enters the spleen and lung meridians. It is a commonly used qi-tonifying herb that invigorates qi, strengthens the spleen, promotes body fluid production, and moistens the lungs. It is suitable for symptoms such as spleen deficiency and fatigue, loss of appetite, weakness after illness, qi and yin deficiency, spontaneous sweating and thirst, and dry cough due to lung dryness.

[0024] Yunzhi: The dried fruiting body of Coriolus versicoLor (L.exFr.)Quel, a fungus belonging to the Polyporaceae family; it is sweet in taste and neutral in nature, and enters the heart, spleen, liver, and kidney meridians; it has the effects of strengthening the spleen and promoting diuresis, clearing heat and detoxifying; it is used for symptoms such as damp-heat jaundice, hypochondriac pain, poor appetite, fatigue, and weakness.

[0025] Licorice: The dried root and rhizome of *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L. (all belonging to the Fabaceae family). It has the effects of tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, alleviating spasms and pain, and harmonizing other herbs. It is commonly used for spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, abdominal and limb spasms and pain, carbuncles and boils, and to alleviate the toxicity and harshness of other drugs.

[0026] (III) Beneficial Effects

[0027] In traditional Chinese veterinary medicine, avian influenza is often classified as a plague disease, characterized by external invasion of pathogenic toxins, lung congestion due to heat toxins, and phlegm obstruction. This invention employs a treatment approach of clearing heat and detoxifying, promoting lung function and resolving phlegm, and supporting the body's resistance to pathogens. A self-formulated traditional Chinese medicine formula has been developed into a herbal feed additive for the prevention and treatment of avian influenza. The formula uses Cordyceps militaris and Belamcanda chinensis as the principal herbs, working synergistically to clear heat and detoxify, dispel wind and pathogens, resolve phlegm and soothe the throat, and promote lung function and resolve stagnation, specifically targeting the pathogenesis of lung congestion due to pathogenic toxins and phlegm-heat obstruction. Arisaema cum Bile clears heat and resolves phlegm, calms wind and relieves convulsions, improving symptoms such as cough and wheezing caused by phlegm obstruction, and enhancing the phlegm-resolving and lung-clearing effects of the principal herbs. Solidago virgaurea clears heat and detoxifies, dispels wind and clears heat, assisting the principal herbs in enhancing the overall effect of the formula in clearing pathogenic heat toxins and dispersing lung heat. These two herbs are considered assistant herbs. Additionally, the formula uses Codonopsis pilosula, which invigorates qi, strengthens the spleen, and moistens the lungs, and Ganoderma lucidum, which invigorates the spleen, invigorates qi, and detoxifies, as adjuvant herbs to jointly achieve the effects of strengthening the body's resistance, consolidating its foundation, and resisting pathogens. Finally, licorice is used as the guiding herb to harmonize the effects of all the herbs. The entire formula is rationally composed of the principal, assistant, adjuvant, and guiding herbs, with all the herbs working synergistically to achieve the effects of clearing heat and detoxifying, promoting lung function and resolving phlegm, and strengthening the body's resistance and eliminating pathogens.

[0028] Based on traditional Chinese veterinary medicine theory, this invention further explores the in vitro antiviral effect of the herbal feed additive. Experimental results show that the herbal feed additive of this invention has no toxic side effects on chicken embryos and exhibits good safety. Under preventative, therapeutic, and inactivation modes of action, the herbal feed additive of this invention can effectively inhibit the proliferation of H9N2 subtype AIV in chicken embryos, demonstrating significant antiviral activity.

[0029] Furthermore, in aquaculture applications, the herbal feed additive of this invention shows significant therapeutic effects on laying hens infected with avian influenza and significantly reduces the mortality rate caused by avian influenza. Simultaneously, the herbal feed additive of this invention can also effectively prevent avian influenza. The herbal feed additive of this invention has enormous application potential in the treatment and prevention of avian influenza. Attached Figure Description

[0030] Figure 1 For the three different modes of action ( Figure 1 A represents a preventative effect. Figure 1 B represents a therapeutic effect. Figure 1 C represents inactivation effect), hemagglutination titers of experimental drug, control drugs 1-2, and OPT; compared with the virus group, *P<0.05, **P<0.01, ***P<0.001; compared with experimental drug, # P<0.05, ## P<0.01. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] A traditional Chinese medicine feed additive for the prevention and treatment of avian influenza is prepared according to the following steps:

[0034] (1) Weigh each raw material according to the following weight parts: 24 parts of Cordyceps militaris, 22 parts of Arisaema cum Bile, 20 parts of Solidago virgaurea, 24 parts of Belamcanda chinensis, 20 parts of Codonopsis pilosula, 20 parts of Ganoderma lucidum, and 8 parts of Glycyrrhiza uralensis. Wash, dry and then pulverize and mix evenly to obtain Chinese medicine raw materials.

[0035] (2) Add 12 times the amount of purified water to the Chinese herbal raw materials, soak for more than 30 minutes, bring to a boil over high heat and keep it at a simmer for more than 2 hours, then filter and separate the first decoction. Add 8 times the amount of purified water to the residue again, bring to a boil over high heat and keep it at a simmer for more than 1.5 hours, then filter and separate the second decoction. Add 6 times the amount of purified water to the residue again, bring to a boil over high heat and keep it at a simmer for more than 1 hour, then filter and separate the third decoction. Combine the three decoctions and refrigerate for 12 hours.

[0036] (3) After standing, filter the decoction, discard the precipitated dregs, collect the clear filtrate, and set aside for later use;

[0037] (4) The clarified filtrate was concentrated under reduced pressure at 60-70℃ and -0.08MPa to -0.1MPa until it became a clear extract with a relative density of 1.15-1.25 at 60℃;

[0038] (5) The extract is spray-dried to make a dry extract powder with a moisture content of less than 5% for later use;

[0039] (6) Anhydrous glucose and soluble starch are mixed at a mass ratio of 2:1.2 as excipients. Then, the dry extract powder is mixed with the excipients at a mass ratio of 1:2. After mixing evenly, the mixture is passed through a 120-150 mesh sieve to obtain the soluble powder of the Chinese herbal feed additive.

[0040] Example 2

[0041] The difference between this embodiment and embodiment 1 is that in step (1), each raw material is weighed according to the following weight parts: 38 parts of Cordyceps militaris, 36 parts of Arisaema cum Bile, 36 parts of Solidago virgaurea, 38 parts of Belamcanda chinensis, 34 parts of Codonopsis pilosula, 32 parts of Ganoderma lucidum, and 18 parts of Glycyrrhiza uralensis.

[0042] Example 3

[0043] The difference between this embodiment and embodiment 1 is that in step (1), each raw material is weighed according to the following weight parts: 28 parts of Cordyceps militaris, 22 parts of Arisaema cum Bile, 24 parts of Solidago virgaurea, 26 parts of Belamcanda chinensis, 22 parts of Codonopsis pilosula, 20 parts of Ganoderma lucidum, and 9 parts of Glycyrrhiza uralensis.

[0044] Example 4

[0045] The difference between this embodiment and embodiment 1 is that in step (1), each raw material is weighed according to the following weight parts: 30 parts of Cordyceps militaris, 25 parts of Arisaema cum Bile, 25 parts of Solidago virgaurea, 30 parts of Belamcanda chinensis, 20 parts of Codonopsis pilosula, 20 parts of Ganoderma lucidum, and 10 parts of Glycyrrhiza uralensis.

[0046] Example 5

[0047] The difference between this embodiment and embodiment 1 is that in step (1), each raw material is weighed according to the following weight parts: 40 parts of Cordyceps militaris, 35 parts of Arisaema cum Bile, 35 parts of Solidago virgaurea, 40 parts of Belamcanda chinensis, 30 parts of Codonopsis pilosula, 30 parts of Ganoderma lucidum, and 16 parts of Glycyrrhiza uralensis.

[0048] Example 6

[0049] The difference between this embodiment and embodiment 1 is that in step (1), each raw material is weighed according to the following weight parts: 34 parts of Cordyceps militaris, 28 parts of Arisaema cum Bile, 30 parts of Solidago virgaurea, 36 parts of Belamcanda chinensis, 24 parts of Codonopsis pilosula, 22 parts of Ganoderma lucidum, and 12 parts of Glycyrrhiza uralensis.

[0050] Example 7

[0051] The difference between this embodiment and embodiment 1 is that in step (1), each raw material is weighed according to the following weight parts: 36 parts of Cordyceps militaris, 30 parts of Arisaema cum Bile, 28 parts of Solidago virgaurea, 32 parts of Belamcanda chinensis, 26 parts of Codonopsis pilosula, 24 parts of Ganoderma lucidum, and 14 parts of Glycyrrhiza uralensis.

[0052] Experimental Example 1

[0053] Evaluation of the in vitro anti-H9N2 avian influenza virus of the herbal feed additive of this invention

[0054] 1. Materials and Methods

[0055] 1.1 Materials

[0056] 1.1.1 Drugs

[0057] Experimental drug: The herbal feed additive of the present invention was prepared according to the method described in Example 7. Before use, it was prepared into a solution with an initial concentration of 200 mg / mL using sterile physiological saline, and then diluted with sterile physiological saline.

[0058] Control Drug 1: The difference between this invention's herbal feed additive and the one prepared in Example 7 is that each herbal ingredient is composed of the following parts by weight: Arisaema cum Bile 30 parts, Solidago virgaurea 28 parts, Codonopsis pilosula 26 parts, Trametes versicolor 24 parts, and Glycyrrhiza uralensis 14 parts. Before use, prepare a solution with an initial concentration of 200 mg / mL using sterile physiological saline, and then dilute it multiple times with sterile physiological saline.

[0059] Control Drug 2: The difference between this invention's herbal feed additive and the one prepared in Example 7 is that each herbal ingredient is composed of the following parts by weight: 36 parts of Cordyceps militaris, 32 parts of Belamcanda chinensis, 26 parts of Codonopsis pilosula, 24 parts of Trametes versicolor, and 14 parts of Glycyrrhiza uralensis. Before use, prepare a solution with an initial concentration of 200 mg / mL using sterile physiological saline, and then dilute it multiple times with sterile physiological saline.

[0060] Oseltamivir phosphate capsules (OTP), 75 mg / capsule, are dissolved in sterile physiological saline to prepare a 1 mg / mL suspension.

[0061] 1.1.2 Seed toxins and chicken embryos

[0062] 10-day-old SPF chicken embryos of H9N2 subtype AIV strain were selected.

[0063] 1.2 Methods

[0064] 1.2.1 Determination of the maximum safe concentration of the herbal feed additive of this invention on chicken embryos

[0065] Take the experimental drug (200 mg / mL), control drug 1 (200 mg / mL), control drug 2 (200 mg / mL), and OTP (1 mg / mL), and respectively dilute them with sterile physiological saline for 2... 1 ~2 3 The four drugs at different concentrations were serially diluted twice and inoculated into 10-day-old SPF-grade chicken embryos via the allantoic cavity. 0.2 mL was injected into each chicken embryo, and 5 chicken embryos were inoculated for each concentration. A blank control group (0.2 mL / embryo) was also set up. The embryos were then incubated in an incubator at (37.5±0.5)℃ and (60±5)% RH. The embryos were observed every 24 hours. The maximum safe concentration of each drug for chicken embryos was determined by the survival of all chicken embryos after 72 hours of incubation.

[0066] 1.2.2 Median Infectivity (EID) of H9N2 subtype AIV in chicken embryos 50 ) Measurement

[0067] Dilute the virus solution with sterile physiological saline to a concentration of 10. -4 Up to 10 -8Then, virus solutions of different concentrations were inoculated into 10-day-old SPF chicken embryos via the allantoic cavity. Each embryo was inoculated with 0.1 mL of virus solution, and five embryos were inoculated for each concentration. The control group was inoculated with 0.2 mL of sterile physiological saline per embryo. The embryos were incubated for 120 hours at (37.5±0.5)℃ and (60±5)% RH. Embryos that died within 24 hours were discarded. The hemagglutination titer of the dead embryos was measured after 24 hours. Embryos with hemagglutination of 4 or more wells were considered positive. The median infection dose (EID) of the chicken embryos was calculated using the Reed-Muench method. 50 ).

[0068] 1.2.3 Activity test of the herbal feed additive of the present invention against H9N2 subtype AIV

[0069] 1.2.3.1 Preventive effect

[0070] Experimental drug (200 mg / mL), control drug 1 (200 mg / mL), control drug 2 (200 mg / mL), and OTP (1 mg / mL) were inoculated into 10-day-old SPF chicken embryos, respectively. Each drug was inoculated into 5 embryos at a rate of 0.2 mL / embryo. After incubation for 2 hours, each embryo was inoculated with 0.1 mL of 100 EID. 50 Virus solution was prepared, with separate control group (0.2 mL sterile physiological saline / embryo) and virus group (0.1 mL 100 EID). 50 Virus fluid / embryo); incubated for 120 h in an incubator at (37.5±0.5) ℃ and (60±5)% RH, and aseptically collected allantoic fluid and determined hemagglutination titer.

[0071] 1.2.3.2 Therapeutic effects

[0072] First, put 100EID 50 Virus solution was inoculated into 10-day-old SPF chicken embryos at a rate of 0.1 mL / embryo. Then, experimental drugs (200 mg / mL), control drug 1 (200 mg / mL), control drug 2 (200 mg / mL), and OTP (1 mg / mL) were inoculated at 0.2 mL / embryo, with each drug inoculated into 5 embryos. A blank control group (0.2 mL / embryo of sterile physiological saline) and a virus group (0.1 mL of 100 EID) were also included. 50 Virus fluid / embryo). Incubated for 120 h in an incubator at (37.5±0.5) ℃ and (60±5)% RH, allantoic fluid was aseptically collected and hemagglutination titer was determined.

[0073] 1.2.3.3 Inactivation effect

[0074] 0.2 mL of the test drug (200 mg / mL), control drug 1 (200 mg / mL), control drug 2 (200 mg / mL), and OTP (1 mg / mL) were respectively mixed with 0.1 mL of 100 EID. 50 The virus solutions were mixed, and after incubation at 4°C for 2 hours, 0.15 mL of each drug mixture was inoculated into 5 chicken embryos. Separate control groups (0.2 mL sterile saline / embryo) and virus groups (0.1 mL 100 EID) were also established. 50 Virus fluid / embryo). Incubate for 120 h in an incubator at (37.5±0.5) ℃ and (60±5)% RH, aseptically collect allantoic fluid and measure hemagglutination titer.

[0075] 1.3 Statistical Methods

[0076] Data were analyzed using GraphPad Prism 8.0.2 software. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and LSD test was used for multiple comparisons. P < 0.05 was considered statistically significant.

[0077] 2 Results

[0078] 2.1 The maximum safe concentration of the herbal feed additive of this invention for chicken embryos

[0079] As shown in Table 1, no chicken embryos in any group died within 72 hours. The maximum safe concentration of the experimental drug and control drugs 1-2 for chicken embryos was 200 mg / mL.

[0080] Table 1. Determination of the maximum safe concentration of the herbal feed additive in chicken embryos according to the present invention.

[0081]

[0082] 2.2 EID of H9N2 subtype AIV 50

[0083] As shown in Table 2, the Reed-Muench method was used to calculate the EID of H9N2 subtype AIV on chicken embryos. 50 10 -5.68 / 0.1mL.

[0084] Table 2. EID of H9N2 subtype AIV 50 Measurement

[0085]

[0086] 2.3 The effect of the herbal feed additive of this invention on the resistance of chicken embryos to H9N2 subtype AIV

[0087] The results are as follows Figure 1As shown, under three different modes of action ( Figure 1 A represents a preventative effect. Figure 1 B represents a therapeutic effect. Figure 1 (C represents inactivation). The hemagglutination titers of the experimental drug, control drugs 1-2, and OPT were all significantly lower than those of the virus group (P<0.001). Under preventive and therapeutic effects, the hemagglutination titer of the experimental drug group was significantly lower than that of control drugs 1-2 (P<0.05, P<0.01), and comparable to that of the OPT group, with no statistically significant difference (P>0.05). Under inactivation effects, the hemagglutination titer of the experimental drug group was lower than that of control drugs 1-2, but the difference was not statistically significant (P>0.05).

[0088] The above results indicate that the herbal feed additive of this invention has no toxic side effects on chicken embryos. Under three different modes of action, the herbal feed additive of this invention can effectively inhibit the proliferation of H9N2 subtype AIV in chicken embryos, demonstrating significant antiviral effects. The various components of the herbal feed additive of this invention complement each other, synergistically enhancing their effects; each component is indispensable.

[0089] Experimental Example 2

[0090] The efficacy of the herbal feed additive of this invention in preventing and treating avian influenza.

[0091] 1. Grouping and Processing

[0092] Forty chickens aged 120-150 days were selected and diagnosed with H9N2 avian influenza based on avian influenza antibody testing and clinical symptoms. Another 120 chickens from the same chicken house that tested negative for avian influenza antibodies and showed no obvious clinical symptoms were selected as the prevention group.

[0093] Clinical symptoms of diseased chickens: green feces, significant decline in mental state and appetite, wheezing, coughing, and shortness of breath, significant decrease in egg production rate, and a significant increase in substandard eggs such as broken eggs, soft eggs, sandy-shelled eggs, and dirty eggs.

[0094] Sick chickens were randomly divided into a control group, experimental group 1 (using the herbal feed additive prepared in Example 4), experimental group 2 (using the herbal feed additive prepared in Example 5), and experimental group 3 (using the herbal feed additive prepared in Example 7), with 100 chickens in each group. The control group received no medication and had free access to feed and water. Experimental groups 1-3 were administered the corresponding herbal feed additives at a dosage of 3g / chick, mixed with water, for 5 days, with free access to feed. The rearing conditions and environment of the laying hens were kept consistent across all groups. After the administration period, the chickens were observed for another 3 days, for a total experimental period of 8 days.

[0095] The laying hens in the prevention group were randomly divided into three groups: Prevention Group 1 (using the herbal feed additive prepared in Example 4), Prevention Group 2 (using the herbal feed additive prepared in Example 5), and Prevention Group 3 (using the herbal feed additive prepared in Example 7), with 40 hens in each group. The dosage was 2g per hen, administered via water, for 5 days. The hens had free access to feed, and the rearing conditions and environment were kept consistent across all groups. After the administration period ended, the hens were observed for another 5 days, for a total experimental period of 10 days.

[0096] 2 Observation Indicators

[0097] 2.1 Criteria for judging the efficacy of treatment in the disease group

[0098] (1) Cured: Within 5 days of the trial period, the feces were normal, the mental state, feed intake and water intake returned to normal, respiratory symptoms such as snoring, coughing and wheezing disappeared, the egg production rate returned to normal, and the rate of unqualified eggs was significantly reduced.

[0099] (2) Effective: During the 8-day trial period, there were no obvious abnormalities in the feces, the mental state, feed intake and water intake recovered significantly, respiratory symptoms such as snoring, coughing and wheezing were significantly reduced, egg production increased and the number of unqualified eggs decreased.

[0100] (3) Invalid: During the 8-day trial period, there were no significant changes in feces, mental state, feed intake, and water intake. Respiratory symptoms such as snoring, coughing, and wheezing did not improve. Egg production continued to decline, the number of unqualified eggs increased, and in severe cases, death occurred.

[0101] 2.2 Criteria for Judging the Prevention Effectiveness of the Prevention Group

[0102] During the trial period, the number of laying hens exhibiting clinical symptoms of avian influenza in each prevention group was observed, and avian influenza antibodies were tested. Successful prevention was defined as the presence of clinical symptoms of avian influenza and a negative avian influenza antibody test, effective prevention was defined as the absence of clinical symptoms of avian influenza but a positive avian influenza antibody test, and prevention failure was defined as the presence of clinical symptoms of avian influenza and a positive avian influenza antibody test.

[0103] 3 Results

[0104] 3.1 The therapeutic effect of the herbal feed additive of this invention on laying hens with avian influenza

[0105] As shown in Table 1, the mortality rate of the diseased laying hens in the blank group without treatment was as high as 78%. After treatment with the herbal feed additive of this invention, the diseased laying hens in each treatment group recovered well, and the total effective rate of treatment was over 98%.

[0106] Table 3. The therapeutic effect of the herbal feed additive of this invention on avian influenza in laying hens [n(%)]

[0107]

[0108] 3.2 The preventive effect of the herbal feed additive of this invention on avian influenza

[0109] As shown in Table 2, the prevention success rate of avian influenza in groups 1-3 all reached over 80%, and the overall prevention effectiveness rate was 97.5%.

[0110] Table 4. The preventive effect of the herbal feed agent of this invention on avian influenza.

[0111]

[0112] In summary, the herbal feed additive of this invention has a significant therapeutic effect on laying hens infected with avian influenza, and can significantly reduce the mortality rate caused by avian influenza. Simultaneously, the herbal feed additive of this invention can also effectively prevent avian influenza. The herbal feed additive of this invention has great application potential in the treatment and prevention of avian influenza.

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine feed additive for the prevention and treatment of avian influenza, characterized in that, The herbal feed additive is made from the following raw materials in parts by weight: 20-40 parts of Cordyceps militaris, 20-40 parts of Arisaema cum Bile, 20-40 parts of Solidago virgaurea, 20-40 parts of Belamcanda chinensis, 20-40 parts of Codonopsis pilosula, 20-40 parts of Ganoderma lucidum, and 5-20 parts of Glycyrrhiza uralensis.

2. The herbal feed additive for preventing and treating avian influenza according to claim 1, characterized in that, The herbal feed additive is made from the following raw materials in parts by weight: 30-40 parts of Cordyceps militaris, 25-35 parts of Arisaema cum Bile, 25-35 parts of Solidago virgaurea, 30-40 parts of Belamcanda chinensis, 20-30 parts of Codonopsis pilosula, 20-30 parts of Ganoderma lucidum, and 10-20 parts of Glycyrrhiza uralensis.

3. The herbal feed additive for preventing and treating avian influenza according to claim 1, characterized in that, The herbal feed additive is made from the following ingredients in parts by weight: 36 parts Cordyceps militaris, 30 parts Arisaema cum Bile, 28 parts Solidago virgaurea, 32 parts Belamcanda chinensis, 26 parts Codonopsis pilosula, 24 parts Ganoderma lucidum, and 14 parts Glycyrrhiza uralensis.

4. The method for preparing a traditional Chinese medicine feed additive for preventing and treating avian influenza as described in claim 1, characterized in that, The herbal feed additive is prepared according to the following steps: (1) Weigh each raw material according to the following weight parts: 20-40 parts of Cordyceps militaris, 20-40 parts of Arisaema cum Bile, 20-40 parts of Solidago virgaurea, 20-40 parts of Belamcanda chinensis, 20-40 parts of Codonopsis pilosula, 20-40 parts of Ganoderma lucidum, and 5-20 parts of Glycyrrhiza uralensis. Wash, dry and then pulverize and mix evenly to obtain the Chinese medicine raw materials. (2) Add 10-14 times the amount of purified water to the Chinese herbal raw materials, soak for more than 30 minutes, bring to a boil over high heat and keep it at a simmer for more than 2 hours, then filter and separate the first decoction. Add 8-10 times the amount of purified water to the residue again, bring to a boil over high heat and keep it at a simmer for more than 1.5 hours, then filter and separate the second decoction. Add 6-8 times the amount of purified water to the residue again, bring to a boil over high heat and keep it at a simmer for more than 1 hour, then filter and separate the third decoction. Combine the three decoctions and refrigerate for 12 hours. (3) After standing, filter the decoction, discard the precipitated dregs, collect the clear filtrate, and set aside for later use; (4) The clarified filtrate was concentrated under reduced pressure at 60-70℃ and -0.08MPa to -0.1MPa until it became a clear extract with a relative density of 1.15-1.25 at 60℃; (5) The extract is spray-dried to make a dry extract powder with a moisture content of less than 5% for later use; (6) Anhydrous glucose and soluble starch are mixed at a mass ratio of 2:1~1.5 as excipients. Then, the dry extract powder is mixed with the excipients at a mass ratio of 1:2~2.

5. After mixing evenly, the mixture is passed through a 120-150 mesh sieve to obtain the soluble powder of the Chinese herbal feed additive.

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