CN111544570B

CN111544570B - Antiviral preparation added with antibacterial peptide and preparation method thereof

Info

Publication number: CN111544570B
Application number: CN202010338529.XA
Authority: CN
Inventors: 刘树军; 屈俊成; 毋会明; 张峰
Original assignee: Diyi Biological Pharmaceutical Co
Current assignee: Diyi Biological Pharmaceutical Co
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2023-05-05
Anticipated expiration: 2040-04-26
Also published as: CN111544570A

Abstract

The invention belongs to the technical field of veterinary medicine preparations, and in particular relates to an antiviral preparation added with antibacterial peptide and a preparation method thereof. The active ingredients of the preparation are prepared from the following components in parts by weight: 5-15 parts of gynostemma pentaphylla, 5-15 parts of astragalus, 5-10 parts of acanthopanax, 5-10 parts of bighead atractylodes rhizome, 5-10 parts of poria cocos, 5-10 parts of liquorice, 5-15 parts of platycodon grandiflorum, 5-10 parts of rheum officinale, 5-10 parts of angelica sinensis, 5-10 parts of magnolia officinalis and 10-15 parts of antibacterial peptide. The preparation used in the invention can effectively strengthen the immunity of organisms through reasonable compatibility. Proved by verification, the anti-virus preparation can improve the content and the stability of the blue-ear disease antibodies in pregnant sows, thereby improving the cure rate of the blue-ear disease; the invention also has obvious treatment effect on infectious bursal disease of chicken.

Description

Antiviral preparation added with antibacterial peptide and preparation method thereof

Technical Field

The invention belongs to the technical field of veterinary medicine preparations, and in particular relates to an antiviral preparation added with antibacterial peptide and a preparation method thereof.

Background

In the livestock industry, the prevention and treatment of viral diseases have plagued farmers, and the banning of human antiviral drugs once causes viral disease outbreaks. The traditional Chinese medicine has great effect on antiviral treatment in the livestock industry, but the traditional Chinese medicine has the limitations of large mixing amount, slow effect and the like, so that the traditional Chinese medicine product which is suitable for the livestock industry and has obvious antiviral effect is an unsolved technical problem at present.

The antibacterial peptide is a kind of micromolecular polypeptide widely existing in organisms and is a main component of natural immunity. The antibacterial peptide has spectral antibacterial activity, can act in a mode of directly killing pathogenic bacteria in vivo, and is used as an immune effector to start and regulate a host immune defense system, such as chemokines of monocytes and T lymphocytes, so that the monocytes and the lymphocytes are rapidly accumulated at an inflammatory reaction site; promoting expression of chemokines and proliferation of T helper lymphocytes; inhibiting bacterial products (such as lipopolysaccharide and lipoteichoic acid) to induce production of cell factor harmful to host, and avoiding endotoxemia; promote apoptosis of macrophages and activated lymphocytes, and has the function of potentially destroying infected cells, etc.

At present, some researches and reports about application of antibacterial peptide to livestock industry are presented, for example, patent CN106359978B discloses a piglet nutrition premix for replacing antibiotics to reduce diarrhea of weaned pigs, which comprises compound probiotics, cecropin antibacterial peptide, plant essential oil, compound enzyme preparation, compound acidulant, organic iron, organic copper, nano zinc oxide, chitosan oligosaccharide, microorganism D3, microorganism C, glutamine and antioxidant, and crushed corn is taken as a carrier; the premix is suitable for the growth and the physiological characteristics of piglets, can replace antibiotics to reduce diarrhea rate of weaned piglets after application, has low cost and is practical in production of compound pig farms. However, the patent product is only aimed at piglets, has the main effect of antidiarrheal and nutrition increasing, and lacks curative effect on other more serious common diseases of animal husbandry, especially infectious diseases.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an antiviral preparation added with antibacterial peptide, and a veterinary antiviral preparation with obvious antiviral curative effect and no influence on food safety is developed by combining a traditional Chinese medicine preparation with the antibacterial peptide; based on one general inventive concept, the invention also comprises a preparation method of the antiviral preparation.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

an antiviral preparation added with antibacterial peptide, wherein the active ingredients of the preparation are prepared from the following components in parts by weight:

5-15 parts of gynostemma pentaphylla, 5-15 parts of astragalus, 5-10 parts of acanthopanax, 5-10 parts of bighead atractylodes rhizome, 5-10 parts of poria cocos, 5-10 parts of liquorice, 5-15 parts of platycodon grandiflorum, 5-10 parts of rheum officinale, 5-10 parts of angelica sinensis, 5-10 parts of magnolia officinalis and 10-15 parts of antibacterial peptide.

Based on one general inventive concept, the invention also comprises a preparation method of the antiviral preparation added with the antibacterial peptide, which comprises the following steps: weighing 10 Chinese medicinal materials except antibacterial peptide according to the formula, and extracting with ethanol; filtering and concentrating the filtrate, adding silicon dioxide and antibacterial peptide with formula amount, mixing, drying and pulverizing.

Preferably, the concentration of ethanol used in the preparation process is 50% -70%.

Preferably, the ethanol extraction is performed for 1-5 hours in two times; extracting for 1-3 hours for the second time; filtering, combining the two filtrates, and concentrating.

Preferably, the temperature of the filtrate is 50-80 ℃ when the filtrate is concentrated; the relative density of the concentrated filtrate is 1.1-1.3.

In the antiviral preparation added with the antibacterial peptide, the main functions of each traditional Chinese medicine are as follows: gynostemma pentaphylla enters lung, spleen and kidney meridians; has effects in invigorating qi, invigorating spleen, eliminating phlegm, relieving cough, and clearing away heat and toxic materials. Astragalus root, radix astragali, having the effects of invigorating qi, raising yang, expelling toxin and expelling pus; mainly enhances the immunity of the organism and improves the immunity. Acanthopanax root, radix acanthopanacis senticosi, capable of replenishing qi to invigorate spleen, tonifying kidney and soothing nerves. Atractylodis rhizoma, bitter and sweet in taste and warm in nature; spleen and stomach meridian; has effects in invigorating spleen, invigorating qi, eliminating dampness, promoting diuresis, and relieving sweat. Poria, having a flavor Gan Danping, entering the heart, lung and spleen meridians; has effects of removing dampness, promoting diuresis, invigorating spleen, regulating stomach function, and tranquilizing mind. Glycyrrhrizae radix is mainly used for clearing heat and detoxicating, eliminating phlegm and relieving cough. Radix Platycodi, ventilating lung, relieving sore throat, eliminating phlegm, and expelling pus; can be used for treating cough with excessive phlegm, chest distress, pharyngalgia, hoarseness, pulmonary abscess, and pyogenic infection. Rhubarb, radix et rhizoma Rhei, having the functions of purging heat, removing the bowels, cooling blood, removing toxin, activating blood stasis and dredging channels. Angelica sinensis, radix angelicae sinensis, capable of replenishing blood, activating blood, regulating menstruation and relieving pain; it also has the actions of moistening dryness and smoothing intestine. Magnolia officinalis, enters spleen meridian, stomach meridian and large intestine meridian; the main functions include promoting qi circulation, removing food retention, eliminating dampness, relieving asthma and lowering adverse qi.

The preparation used in the invention can effectively strengthen the immunity of organisms through reasonable compatibility. Proved by verification, the anti-virus preparation can improve the content and the stability of the blue-ear disease antibodies in pregnant sows, thereby improving the cure rate of the blue-ear disease; the invention also has obvious treatment effect on infectious bursal disease of chicken. Analysis of the T lymphocyte level of the piglets after taking the preparation shows that the antiviral preparation can improve the T lymphocyte level of the piglets and maintain the T lymphocyte content in a higher range for a long time, thereby improving the disease resistance of the bodies of the piglets.

The beneficial effects of the invention are as follows:

1) The obtained antiviral preparation has good therapeutic effect on various viral infectious diseases in livestock and fowl cultivation;

2) The effective components in the product are fully extracted by adopting a scientific traditional Chinese medicine extraction process, and the obtained preparation is easy to administer and is beneficial to animal absorption;

3) The preparation does not contain antibiotics and other intermediate products which are not easy to metabolize, does not bring hidden danger to downstream food safety, and is a green and efficient veterinary antiviral pharmaceutical preparation.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments.

The antibacterial peptide used in the invention is peptide hormone 727C (Shandong Baolier bioengineering Co., ltd.); other medicinal materials and preparations are all commonly commercially available or available to those skilled in the art through public routes.

Examples

Weighing 3000g of 10 traditional Chinese medicines of gynostemma pentaphylla, astragalus, acanthopanax, bighead atractylodes rhizome, poria cocos, liquorice, platycodon grandiflorum, rheum officinale, angelica sinensis and magnolia officinalis, and extracting twice with 50% ethanol at room temperature: extracting with 50% ethanol for 2 hr, and separating solid from liquid; adding 50% ethanol into the residue again, and extracting for 1 hr; filtering; combining the two filtrates; concentrating the filtrate at 60deg.C under reduced pressure to relative density of 1.2; then adding a certain amount of antibacterial peptide and silicon dioxide into the concentrated solution, wherein the addition amount of the silicon dioxide is calculated by supplementing the mass of the finally obtained preparation to 1000 g; mixing uniformly; drying at 80deg.C, and pulverizing.

The amounts of the traditional Chinese medicines and the antibacterial peptides used in the different examples are shown in Table 1;

table 1 proportions (units: parts) of the respective components in the different embodiments

The formulation of the traditional Chinese medicine composition of the above example 4 is the same as that of examples 1-3, but no antibacterial peptide is added. The inventors performed the following pharmacological analyses with respect to the antiviral preparations obtained in examples 1 to 4.

Test example 1

In order to examine the effect of the antiviral preparation of the present invention after being applied to animals, the inventors first tested the therapeutic effect of the above-mentioned agents on porcine reproductive and respiratory syndrome. Blue-ear disease is a highly contagious disease, and is characterized by reduced productivity of herd, slow growth, reduced reproductive performance of herd, reduced immune function of herd, susceptibility to secondary infection with other bacterial and viral diseases, with pregnant sows and piglets within 1 month of age being most susceptible.

1) Pharmaceutical agents

Test group I, group II, group III: the antiviral preparations prepared in examples 1, 2 and 3;

control group i: the pharmaceutical formulation prepared in example 4;

control group ii: antipyretic and antitoxic powder (prepared according to pharmacopoeia).

2) Test method

According to the principle of identical variety and equivalent weight, 100 pregnant sows are randomly selected, and 20 pregnant sows are tested in groups I, II and III and 20 pregnant sows are tested in groups I and II. The usage amounts of the test group I, the test group II, the test group III and the control group I are as follows: 1000kg of water is added into 1000g of medicament to prepare an aqueous solution, and the aqueous solution is used for 7 days along with pregnant sow drinking water. The dosage of the control II group medicine is as follows: mixed feeding, each pig is 100g per day for 7 days.

3) Test results and analysis

The therapeutic effects of different medicaments on the blue ear disease of pregnant sows are shown in table 2;

table 2 comparison of therapeutic effects of different agents administered to pregnant sow against blue ear disease

As can be seen from table 2, the effective rates of administration of the test group agents for treating blue ear disease of pregnant sows were 85%, 85% and 90%, respectively; the cure rate is 65%, 65% and 70% respectively, which are all higher than the control group medicament; among them, the therapeutic effect of the agent obtained in example 3 was optimal.

In order to further reveal the specific reason that the medicament is effective for the blue-ear disease, the ELISA antibody detection method is utilized to detect the in vivo blue-ear disease antibody level of the sow in the test group and the control group respectively; specific detection results are shown in Table 3;

TABLE 3 comparison of blue ear disease antibody levels in pregnant sows under different drug administration conditions

Note that: the same letter for the same column data shoulder indicates that the difference is not significant (P > 0.05).

In the above table, an S/P value of 0.4 or more indicates positive and an S/P value of <0.4 indicates negative. As can be seen from the above table, the S/P values of the above five groups of test conditions are all >0.4, which indicates that the blue ear virus antibodies are produced in the pregnant sows of the above five groups.

In addition, the data in the tables show that the S/P values 0-2 represent stable antibody levels, the S/P values 0.7-1.8 represent good antibody levels, and the S/P values >2.0 represent unstable antibody levels. It can be seen that the stable antibody level ratios of the test group I, the test group II and the test group III are 89.3%, 90.8% and 89.9%, respectively, which are higher than the control group I and the control group II, namely, 51.3% and 42.9%, respectively, which shows that the administration of the medicaments prepared in the examples 1-3 can obviously improve the antibody level stability of the pregnant sow blue-ear disease; the unstable levels of antibodies in groups I, II and III were found to be 10.7%, 9.2% and 10.1%, respectively, less than 48.7% for control group I and 57.1% for control group II, indicating that administration of examples 1-3 was effective in reducing the antibodies to blue ear disease in pregnant sows.

The data result shows that compared with the traditional antiviral preparation, the preparation (example 4) obtained by adopting the compatibility of the traditional Chinese medicines can improve the antibody level of the blue ear disease of pregnant sows to a certain extent, thereby improving the cure rate of the blue ear disease, but improving the cure rate to a smaller extent; and after the antibacterial peptide is added (examples 1-3), the antibody level of the blue-ear disease of pregnant sows can be improved, the stability of the antibody can be enhanced, and finally the cure rate of the blue-ear disease is obviously improved.

Test example 2

The inventors next tested the therapeutic effect of the above agents on infectious bursal disease in chickens. Infectious Bursal Disease (IBD), also known as Gan Boluo disease, is an acute, highly infectious disease caused by infectious bursal virus. The disease has sudden onset, short course and high death rate, can cause chicken immunosuppression, and is one of main infectious diseases in chicken raising industry at present.

1) Pharmaceutical agents

The same test agent as in test example 1 was used.

2) Test method

According to the principle of identical variety and equivalent weight, 500 chickens (25 days old) are randomly selected, and 100 feathers of the test group I, the test group II and the test group III and the control group I and the control group II are tested. The usage amounts of the test group I, the test group II, the test group III and the control group I are as follows: 1000kg of water is added into 1000g of the medicament to prepare an aqueous solution, and the aqueous solution is taken along with chicken drinking water for 7 days. The dosage of the control II group medicine is as follows: mixed feeding, each chicken is 2g, and the mixture is taken for 7 days.

3) Test results and analysis

The therapeutic effects of different medicaments on infectious bursal disease of chickens are shown in Table 4;

TABLE 4 comparison of therapeutic effects of different agents administered on infectious bursal disease in chickens

As can be seen from table 4, the treatment effect on infectious bursal disease of chicken after taking the medicaments of test 1-3 groups is obviously better than that of the control group; wherein the cure rate of bursal disease in test 1 group is 71%, the death rate is 5%, the total effective rate reaches 89%, and the overall treatment effect is optimal. The traditional Chinese medicine preparation without the antibacterial peptide added in the control group I has the cure rate of 45 percent, the death rate of 11 percent and the total effective rate of 66 percent for the infectious bursal disease of chicken, and the treatment effect is slightly higher than that of the control group II.

Test example 3

To further reveal the cause of the effect of the agent of the present invention on the above diseases, the inventors tested the change in the T lymphocyte content of the body after taking the agent in a pig.

Randomly selecting 40 weaned pigs, keeping the variety, the day age and the weight consistent, and testing 10 groups I, II and III; the dosage of the test group medicament is as follows: 1000kg of water is added into 1000g of the medicament to prepare an aqueous solution, and the aqueous solution is used for drinking with piglets. Taking weaned piglets without feeding medicaments as a blank control group, wherein the number of the weaned piglets is 10;

blood was collected 1, 4, 25, 40, 50, and 60 days after administration and before feeding in the morning, respectively, and a blood smear was prepared. Staining with acid alpha-acetate (ANAE) staining method, and microscopic examination of ANAE+T lymphocytes under an optical microscope. The presence of reddish brown particles in the cytoplasm was noted as tANAE+T cells, whereas none; the ratio of the number of T cells to the total cells in the field is taken as the content. The results of T lymphocyte microscopic examination of piglets under different administration conditions are shown in Table 5;

table 5 comparison of piglet T lymphocyte assay results (%)

As can be seen from the data in Table 5, the T cell content of piglets in the test group I, the test group II and the test group III are higher than those in the control group. And when the medicine is taken for 40-60 days, the T cell content of the piglet is not reduced but is increased to a certain extent. This demonstrates that administration of the antiviral agents of examples 1-3 increases T lymphocyte levels in pigs and maintains the immune levels at high levels for a longer period of time, thereby increasing the resistance of the piglet's body to disease. In addition, from 60 sky white data, the T cell level in the piglet body is influenced by external environment or feeding conditions and is relatively stable after administration, so that the organism immunity level is in a relatively stable state and the piglet resistance is improved.

Claims

1. An antiviral preparation added with antibacterial peptide is characterized in that the active ingredients of the preparation are prepared from the following components in parts by weight:

5-15 parts of gynostemma pentaphylla, 5-15 parts of astragalus, 5-10 parts of acanthopanax, 5-10 parts of bighead atractylodes rhizome, 5-10 parts of poria cocos, 5-10 parts of liquorice, 5-15 parts of platycodon grandiflorum, 5-10 parts of rheum officinale, 5-10 parts of angelica sinensis, 5-10 parts of magnolia officinalis and 10-15 parts of antibacterial peptide, wherein the antibacterial peptide is peptide bacteriocin 727C.

2. The method for preparing an antibacterial peptide-added antiviral preparation according to claim 1, wherein: weighing 10 Chinese medicinal materials except antibacterial peptide according to the formula, and extracting with ethanol; filtering and concentrating the filtrate, adding silicon dioxide and antibacterial peptide with formula amount, mixing, drying and pulverizing.

3. The method of manufacturing as claimed in claim 2, wherein: the concentration of ethanol used is 50% -70%.

4. A method of preparation as claimed in claim 3, wherein: the ethanol extraction is carried out twice, and the first extraction is carried out for 1-5 hours; extracting for 1-3 hours for the second time; filtering, combining the two filtrates, and concentrating.

5. The method of any one of claims 2-4, wherein: the temperature of the filtrate is 50-80 ℃ when the filtrate is concentrated; concentrating the filtrate to a relative density of 1.1-1.3.