CN111544570A

CN111544570A - Antiviral preparation added with antibacterial peptide and preparation method thereof

Info

Publication number: CN111544570A
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: 2020-08-18
Anticipated expiration: 2040-04-26
Also published as: CN111544570B

Abstract

The invention belongs to the technical field of veterinary medicine preparations, and particularly relates to an antiviral preparation added with antibacterial peptide and a preparation method thereof. The effective components of the preparation are prepared from the following components in parts by weight: 5-15 parts of gynostemma pentaphylla, 5-15 parts of astragalus membranaceus, 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 mangnolia officinalis and 10-15 parts of antibacterial peptide. The preparation used by the invention can effectively enhance the immunity of the organism through reasonable compatibility. Proved by verification, the antiviral preparation can improve the content and stability of the blue-ear disease antibody in the pregnant sow body, thereby improving the cure rate of the blue-ear disease; the invention also has obvious treatment effect on the chicken infectious bursal disease.

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 particularly relates to an antiviral preparation added with antibacterial peptide and a preparation method thereof.

Background

In the animal husbandry industry, the prevention and treatment of viral diseases always troubles farmers, and the viral diseases are once outbreaked when antiviral drugs for human use are forbidden. The traditional Chinese medicine has a huge effect on antiviral treatment in the livestock industry, but the use of the traditional Chinese medicine has limitations, such as large mixing amount, slow effect and the like, so that the research and development of traditional Chinese medicine products which are suitable for the livestock industry and have obvious antiviral effect is a technical problem which is not solved at present.

The antibacterial peptide is a small molecular polypeptide widely existing in organisms and is a main component of natural immunity. The antibacterial peptide has spectrum antibacterial activity, can act in a mode of directly killing pathogenic bacteria in vivo, and can be used as an immune effector molecule to start and regulate a host immune defense system, such as acting as a chemotactic factor of monocytes and T lymphocytes, so that the monocytes and the lymphocytes are rapidly gathered at an inflammation reaction part; promoting the expression of chemical factors and the proliferation reaction of T helper lymphocytes; inhibiting bacterial products (such as lipopolysaccharide and lipoteichoic acid) from inducing and producing cell factors harmful to a host, and avoiding endotoxemia; promoting the apoptosis of macrophages and activated lymphocytes, and having the potential function of eliminating infected cells.

At present, some research reports about the application of antibacterial peptides in the animal husbandry industry exist, for example, patent CN106359978B discloses a piglet nutrition premix for replacing antibiotics to reduce diarrhea of weaned piglets, which comprises composite probiotics, cecropin antibacterial peptides, plant essential oil, a composite enzyme preparation, a composite acidifier, organic iron, organic copper, nano zinc oxide, chitosan oligosaccharide, microorganism D3, microorganism C, glutamine and an antioxidant, wherein crushed corn is used as a carrier; the premix is suitable for the growth, development and physiological characteristics of piglets, can replace antibiotics to reduce the diarrhea rate of weaned piglets after application, and has low cost and practical production of a compound pig farm. However, the product only aims at piglets, has the main effects of relieving diarrhea and increasing nutrition, and has no treatment effect on other more serious common diseases in 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 the antiviral preparation for animals, which has obvious antiviral curative effect and does not influence food safety, is developed by combining a traditional Chinese medicine preparation and the antibacterial peptide; based on a general inventive concept, the present invention also includes a method for preparing the antiviral preparation.

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

an antiviral preparation added with antibacterial peptide comprises the following effective components in parts by weight:

5-15 parts of gynostemma pentaphylla, 5-15 parts of astragalus membranaceus, 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 mangnolia officinalis and 10-15 parts of antibacterial peptide.

Based on a general inventive concept, the present invention also includes a method for preparing the antiviral preparation with the antibacterial peptide added, which comprises the following steps: weighing 10 Chinese medicinal materials except the antibacterial peptide according to the formula ratio, and extracting with ethanol; filtering and concentrating the filtrate, then adding silicon dioxide and the antibacterial peptide with the formula amount, uniformly mixing, drying and crushing to obtain the antibacterial peptide.

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

Preferably, the ethanol extraction is carried out twice, and the first extraction is carried out for 1-5 hours; extracting for 1-3 h for the second time; filtering, combining the two filtrates, and concentrating.

Preferably, the temperature of the filtrate during concentration is 50-80 ℃; 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 the traditional Chinese medicines are as follows: gynostemma pentaphyllum Makino enters lung, spleen and kidney meridians; has the effects of invigorating qi, strengthening spleen, eliminating phlegm, relieving cough, and clearing away heat and toxic materials. Astragalus root, radix astragali, invigorating qi, invigorating yang, expelling toxin, and expelling pus; mainly enhances the immune function of the organism and improves the immune function. Acanthopanax senticosus has the effects of tonifying qi, invigorating spleen, tonifying kidney and soothing nerves. Bighead atractylodes rhizome, bitter in taste, sweet and warm in nature; spleen and stomach meridians entered; has the effects of invigorating spleen, invigorating qi, eliminating dampness, promoting diuresis, and stopping sweating. Poria, sweet and mild in nature and flavor, enters heart, lung and spleen meridians; has effects of eliminating dampness, promoting diuresis, invigorating spleen, regulating stomach function, calming heart, and tranquilizing mind. Licorice root, radix Glycyrrhizae is indicated for clearing away heat and toxic material, expelling phlegm and arresting cough. Balloonflower root, radix Platycodi, has effects of dispersing lung qi, relieving sore throat, eliminating phlegm, and expelling pus; can be used for treating cough with excessive phlegm, chest distress, pharyngalgia, hoarseness, lung carbuncle, suppuration, and pyocutaneous disease with pus formation. Rhubarb, radix et rhizoma Rhei, purges heat and unblocks intestines, cools blood and detoxifies, promotes blood stasis and dredges the channels. The angelica sinensis can enrich the blood, promote blood circulation, regulate menstruation and relieve pain; has the effects of moistening dryness and smoothing intestine. Magnolia officinalis, entering spleen channel, stomach channel and large intestine channel; the main functions include promoting qi circulation, removing food retention, eliminating dampness, relieving fullness, lowering adverse qi and relieving asthma.

The preparation used by the invention can effectively enhance the immunity of the organism through reasonable compatibility. Proved by verification, the antiviral preparation can improve the content and stability of the blue-ear disease antibody in the pregnant sow body, thereby improving the cure rate of the blue-ear disease; the invention also has obvious treatment effect on the chicken infectious bursal disease. Analysis of the T lymphocyte level of the piglets after the medicine is taken shows that the antiviral preparation can improve the T lymphocyte level of the pigs when the antiviral preparation is taken, and can maintain the T lymphocyte content in a higher range for a longer time, thereby improving the disease resistance of the piglet organisms.

The invention has the beneficial effects that:

1) the obtained antiviral preparation has good therapeutic effect on various viral infectious diseases in livestock breeding;

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 the absorption of animals;

3) the preparation does not contain antibiotics and other intermediates which are difficult to metabolize, does not bring hidden danger to the safety of downstream food, and is a green and efficient veterinary antiviral medicinal preparation.

Detailed Description

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

The antibacterial peptide used in the invention is peptin 727C (Shandong Baoli biological engineering Co., Ltd.); other medicinal materials and preparations are all generally sold in the market or can be obtained by the public way of the technical personnel in the field.

Examples

Weighing 3000g of 10 traditional Chinese medicines including gynostemma pentaphylla, astragalus, acanthopanax, bighead atractylodes rhizome, poria cocos, liquorice, platycodon grandiflorum, rheum officinale, angelica and mangnolia officinalis, and extracting with 50% ethanol at room temperature for two times: adding 50% ethanol, extracting for 2 hr, and performing solid-liquid separation; adding 50% ethanol into the filter residue again, and extracting for 1 hour; filtering; mixing the two filtrates; concentrating the filtrate at 60 deg.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 adding amount of the silicon dioxide is calculated by complementing the mass of the final preparation to 1000 g; mixing uniformly; drying at 80 deg.C, and pulverizing.

The dosage proportion of the traditional Chinese medicines and the antibacterial peptide used in different embodiments is shown in table 1;

TABLE 1 proportioning of the components in different embodiments (unit: parts)

The formulation of the herbal composition of example 4 above is the same as examples 1-3, but without the addition of antimicrobial peptides. The inventors conducted the following pharmacological analysis with respect to the antiviral preparations obtained in examples 1 to 4.

Test example 1

In order to examine the effect of the antiviral agent of the present invention after it is applied to animals, the inventors first tested the therapeutic effect of the above agent on porcine reproductive and respiratory syndrome. The blue ear disease is a highly contagious disease, and is characterized by the reduction of the productive performance of a pig group, slow growth, the reduction of the reproductive performance of a sow group, the reduction of the immune function of the pig group and easy secondary infection of other bacterial and viral diseases after infection, wherein pregnant sows and piglets within 1 month of age are most susceptible.

1) Pharmaceutical agent

Test group i, group ii, group iii: the antiviral formulations prepared in examples 1, 2, 3;

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

control group ii: scourge-clearing toxin-vanquishing powder (prepared according to pharmacopoeia).

2) Test method

According to the principle of same variety and equal weight, 100 pregnant sows are randomly selected, and 20 sows in the groups I, II and III of the test and the groups I and II of the control are selected. The usage and dosage 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 aqueous solution, and the aqueous solution is used along with pregnant sows for 7 days. The usage and dosage of the medicines in the control group II are as follows: the feed is mixed, each pig is 100g per day, and the feed is continuously used for 7 days.

3) Test results and analysis

The treatment effect of taking different medicaments on the porcine reproductive and respiratory syndrome of pregnant sows is shown in table 2;

TABLE 2 comparison of the therapeutic effects of different drugs on porcine reproductive and respiratory syndrome in pregnancy

As can be seen from the table 2, the treatment effective rates of the medicines taken in the test group on the porcine reproductive and respiratory syndrome of pregnant sows are 85%, 85% and 90% respectively; the cure rates are respectively 65%, 65% and 70%, which are all higher than the control group medicament; among them, the therapeutic effect was the most excellent with the agent obtained in example 3.

In order to further reveal the specific reason that the medicament is effective to the blue-ear disease, the in-vivo blue-ear disease antibody levels of sows of a test group and a control group are respectively detected by an ELISA antibody detection method; the specific detection results are shown in table 3;

TABLE 3 comparison of pregnant sow blue-ear disease antibody levels under different drug-taking conditions

Note: the data in the same column are shoulder marked with the same letter to indicate that the difference is not significant (P > 0.05).

In the above table, an S/P value of 0.4 or more indicates positive antibody, and an S/P value of 0.4 or less indicates negative antibody. As can be seen from the table above, the S/P values of the above five groups of experimental conditions were all >0.4, indicating that the above five groups of pregnant sows all produced PRRSV antibodies in vivo.

In addition, the data in the table indicate that the antibody levels are stable at S/P values of 0-2, good at S/P values of 0.7-1.8, and unstable at S/P values > 2.0. It can be seen that the stable antibody levels 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 51.3% of the control group I and 42.9% of the control group II, which shows that the stability of the pregnant sow blue-ear disease antibody levels can be significantly improved by taking the medicament prepared in the examples 1-3; the unstable ratio of the antibody levels of the test group I, the test group II and the test group III is 10.7%, 9.2% and 10.1%, respectively, which is lower than 48.7% of the control group I and 57.1% of the control group II, which shows that the intake of the example 1-3 can effectively reduce the unstable antibody level of the pregnant sow blue ear disease.

The data result shows that compared with the traditional antiviral preparation plague-clearing toxin-vanquishing powder, the preparation prepared by adopting the compatibility of the traditional Chinese medicines (example 4) can improve the antibody level of the porcine reproductive and respiratory syndrome of pregnant sows to a certain extent, so that the cure rate of the porcine reproductive and respiratory syndrome is improved, but the improvement degree is smaller; after the antibacterial peptide is added (examples 1-3), the antibody level of the porcine reproductive and respiratory syndrome of the pregnant sow can be improved, the stability of the antibody can be enhanced, and the cure rate of the porcine reproductive and respiratory syndrome can be obviously improved finally.

Test example 2

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

1) Pharmaceutical agent

The same test agents as in test example 1 were used.

2) Test method

According to the principle of same variety and equal weight, 500 chickens (25 days old) are randomly selected, and 100 feathers are respectively selected from a test group I, a test group II and a test group III and a control group I and a control group II. The usage and dosage 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 aqueous solution, and the aqueous solution is taken with chickens for 7 days. The usage and dosage of the medicines in the control group II are as follows: the feed is mixed, 2g is taken by each chicken for 7 days.

3) Test results and analysis

The treatment effect of taking different medicaments on the infectious bursal disease of chicken is shown in table 4;

TABLE 4 comparison of the therapeutic effects of different drugs on chicken infectious bursal disease

As can be seen from Table 4, the treatment effect of the test groups 1-3 on the infectious bursal disease after the drug is taken is obviously better than that of the control group; wherein, the cure rate of the bursal disease of the test 1 group is 71 percent, the death rate is 5 percent, the total effective rate reaches 89 percent, and the total treatment effect is optimal. The traditional Chinese medicine preparation without the antibacterial peptide 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 on the infectious bursal disease of chicken, and the treatment effect is slightly higher than that of the control group II.

Test example 3

In order to further reveal the cause of the effect of the agent of the present invention on the above-mentioned diseases, the inventors tested the change in T lymphocyte content in the body after administration to pigs.

Randomly selecting 40 weaned piglets, keeping the variety, the age in days and the weight consistent, and testing 10 weaned piglets in the I group, the II group and the III group respectively; the using method and dosage of the test group medicament are as follows: 1000kg of water is added into 1000g of the medicament to prepare aqueous solution which is used along with drinking water for piglets. Weaned piglets which are not fed with the medicament are used as a blank control group, and the number of the piglets is 10;

blood is collected before feeding on 1, 4, 25, 40, 50 and 60 morning after administration, and blood smear is prepared. Staining with acid alpha-acetate esterase (ANAE) staining method, and performing microscopic examination on ANAE + T lymphocytes under an optical microscope. The presence of reddish-brown particles in the cytoplasm was scored as tANAE + T cells, whereas none was present; the content is given as the percentage of the number of T cells to the total cells in the field. The results of the T lymphocyte microscopic examination of the piglets under different medicine taking conditions are shown in the 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 groups I, II and III is higher than that in the control group. And when the medicine is taken for 40-60 days, the T cell content of the piglets is not reduced but increased to a certain extent. This indicates that the administration of the antiviral agents of groups 1 to 3 of examples can increase the level of T lymphocytes in pigs and maintain the immune level of the body at a high level for a long time, thereby improving the disease resistance of the body of piglets. In addition, as can be seen from the data of 60 sky white group, the T cell level in the piglet body is greatly fluctuated under the influence of external environment or feeding conditions, and the T cell level in the test group is relatively stable after the medicine is taken, so that the body immunity level is in a relatively stable state, and the piglet resistance is improved.

Claims

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

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

3. The method of claim 2, wherein: the concentration of the ethanol is 50-70%.

4. The method of 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 h for the second time; filtering, combining the two filtrates, and concentrating.

5. The production method according to any one of claims 2 to 4, characterized in that: the temperature of the filtrate during concentration is 50-80 ℃; the filtrate is concentrated to a relative density of 1.1-1.3.