Veterinary antiviral medicine powder and preparation method thereof

Abstract

The invention relates to the field of veterinary drugs, and particularly discloses veterinary antiviral medicine powder and a preparation method thereof.The veterinary antiviral medicine powder is prepared from, by weight, 60-68 parts of auxiliary materials, 28-34 parts of nano-medicine and 4-7 parts of compound accelerant, the encapsulation efficiency of the nano-medicine is larger than or equal to 80.0%, and the drug loading capacity is larger than or equal to 5.0%; according to the preparation method, algal polysaccharides with negative charges are adsorbed on the surface of chitosan with positive charges through electrostatic adsorption, and then the algal polysaccharides and the chitosan are polymerized with sodium tripolyphosphate to obtain the nano-drug loaded with vidarabine, so that the antiviral activity of the antiviral drug in an animal body is improved, and the toxicity to the animal body is reduced while the dosage is reduced; and the delivery and the stability of the nano-drug in a living body are improved by compounding the accelerant.

Description

Technical Field

[0001] The invention belongs to the field of veterinary medicines, and particularly relates to an antiviral medicine powder for animals and a preparation method thereof. Background Art

[0002] Intensive livestock and poultry farming is the main trend of the current animal husbandry industry. This intensive farming method reduces production costs, but also brings the risk of large-scale viral disease outbreaks. Veterinary antiviral drugs mainly work by inhibiting viral replication or enhancing host immunity in clinical practice. Common veterinary antiviral drugs include interferon, adenosine, ribavirin, acyclovir and famciclovir. When treating livestock and poultry diseases, due to lack of professional knowledge, when the economic and time costs are high, the breeding entities generally adopt the strategy of "better to be safe than sorry" to prevent suspected sick livestock and poultry. Although these veterinary antiviral drugs play a strong role in preventing and treating animal viral infections, long-term use of antiviral drugs may lead to viral mutations and drug resistance. If the drug resistance problem develops further, the breeding entities will lose the means to effectively treat and prevent animal diseases; and the irrational use of these antiviral drugs in poultry and livestock may also affect human health.

[0003] In order to reduce the use of antiviral drugs and ensure their safety and effectiveness at all levels, many countries encourage the development of alternative drugs. my country strongly supports the development of Chinese herbal veterinary drugs to replace chemical veterinary antiviral drugs. For example, patent CN104383068B discloses a highly effective antiviral and antibacterial Chinese veterinary drug, which uses pure natural Chinese herbal medicines and achieves antiviral effects through the combination of components, and has no toxic side effects on livestock and poultry bodies. Patent CN111544570B discloses an antiviral preparation with added antimicrobial peptides and a preparation method thereof, which can treat blue ear disease and infectious bursal disease in chickens, and improves the antiviral efficacy by adding antimicrobial peptides to Chinese medicine without affecting food safety. However, both of the above have the limitation that Chinese medicine takes effect slowly, which makes the antiviral effect not significant when facing highly contagious and fast-onset viruses.

[0004] Therefore, there is an urgent need on the market for a veterinary antiviral drug that is fast-acting and requires little dosage. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention prepares antiviral components into nanomedicines to improve their antiviral activity in animals. The antiviral activity of the nanomedicines includes seaweed polysaccharides and adenosine; at the same time, the delivery and stability of the nanomedicines in the organism are improved by compounding promoters, thereby obtaining an antiviral powder for veterinary use which has a fast onset of action, a small dosage and low toxicity to animals.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides an antiviral powder for veterinary use, which comprises the following raw materials, by weight: 60 to 68 parts of auxiliary materials, 28 to 34 parts of nano drugs and 4 to 7 parts of compounding promoters; wherein the encapsulation rate of the nano drugs is ≥80.0%, and the drug loading is ≥5.0%.

[0008] In some embodiments, the preparation steps of the nanomedicine are as follows:

[0009] S1. Dissolve chitosan in acetic acid solution at room temperature, stir until there is no solid, adjust the pH to 4.6-5.4, add adenosine, stir for 10-15 minutes, then add EDC and NHS and stir for 100-150 minutes, add anhydrous ethanol after stirring until no precipitation, centrifuge to obtain the supernatant, and freeze-dry to obtain a chitosan drug complex;

[0010] S2, adding the seaweed polysaccharide solution to the chitosan drug complex obtained in step S1, stirring at room temperature for 25 to 35 minutes, adding the sodium tripolyphosphate solution, continuing to stir for 25 to 35 minutes, adding poloxamer 188, continuing to stir for 50 to 70 minutes, centrifuging to remove the precipitate, and freeze-drying to obtain the nano drug.

[0011] For livestock and poultry, oral administration is the preferred form of application for most drugs, but the oral absorption efficiency of drugs is affected by the physical and chemical properties of the drugs and the physiological factors of the person taking the drugs. Since neither viruses nor animal cells have cell walls, when developing antiviral drugs, it is impossible to study the cell walls as targets like antibacterial drugs, resulting in most antiviral drugs being unable to fully identify the target of action. In order to achieve the efficacy, the drugs are generally used in large quantities, and it is difficult to avoid toxicity to animal cells.

[0012] Adenosine is a purine nucleoside homologue that can block viral synthesis by inhibiting viral DNA polymerase. It is mainly used in veterinary clinics to prevent and treat pseudorabies in piglets, keratitis caused by herpes simplex virus, bovine papillomavirus damage and equine herpes virus. The nanomedicine provided by the present invention can improve the antiviral activity of adenosine in animals while reducing its toxicity to the animals themselves. The specific principles are as follows:

[0013] First, seaweed polysaccharides have certain antiviral effects, but seaweed polysaccharides have a large molecular weight and contain a large number of polar groups, making it difficult to be absorbed through the gastrointestinal mucosa into the blood circulation, and they are easily inactivated by metabolism in the body and lose their antiviral effects. The present invention is based on the principle of electrostatic adsorption, adsorbs negatively charged seaweed polysaccharides on the surface of positively charged chitosan, and then prepares nanomedicines containing seaweed polysaccharides by polymerizing with sodium tripolyphosphate, thereby extending the action time of seaweed polysaccharides and thereby improving the antiviral activity of seaweed polysaccharides;

[0014] Secondly, since the paracellular pathway is restricted by tight junctions (TJs), adhesion junctions and desmosome protein complexes between adjacent epithelial cells when drug molecules are transported through transcellular / intercellular mechanisms, the nanostructure of the nanomedicine of the present invention can trigger the opening of TJs and promote the transport of drugs through epithelial cells, and the small structure of the nanomedicine is conducive to increasing the concentration of the drug in the virus aggregation area;

[0015] Thirdly, the chitosan in the nanomedicine of the present invention increases its residence time in the mucus layer of the gastrointestinal tract; and the sulfate groups contained in seaweed polysaccharides can not only reduce the degree of mucus cross-linking, but also form disulfide bonds in mucin, thereby enhancing the adhesion ability of the nanomedicine, and the two synergistically increase the drug permeability.

[0016] In some embodiments, in step S1, the mass ratio of chitosan to adenosine is 1:(0.03-0.07).

[0017] In some embodiments, in step S2, the concentration of the seaweed polysaccharide solution is 0.3-0.8 mg / mL, and the concentration of the sodium tripolyphosphate solution is 0.1-0.3 mg / mL.

[0018] In some embodiments, the ratio of chitosan in step S1 to the seaweed polysaccharide solution in step S2 is 1 mg: (0.2-0.3) mL.

[0019] In some embodiments, the ratio of chitosan in step S1 to the sodium tripolyphosphate solution in step S2 is 1 mg: (0.45-0.55) mL.

[0020] In the process of preparing the nano drug, the invention ensures the encapsulation rate and drug loading of adenosine in the nano drug by specifically selecting the dosage of the raw materials.

[0021] In some embodiments, the compounding promoter comprises Tween-20 and alkyl glycoside.

[0022] In some embodiments, the mass ratio of Tween-20 to alkyl glycoside is (0.33-0.45):1.

[0023] Since the nano drug of the present invention contains sodium tripolyphosphate, charge reversal may occur under enzyme triggering, resulting in reduced stability of the nano drug in vivo, making it easy to disintegrate and unable to effectively play the role of the nano structure. The present invention improves the delivery and stability of the nano drug by compounding the promoter, wherein Tween-20 can reduce the hydrophobic interaction between enzymes, thereby reducing the adsorption of enzymes on the surface of the nano drug and improving the stability of the nano drug; and alkyl glycoside can instantly and reversibly loosen the tight connection between cells and change the viscosity of the mucosa, promoting the penetration of drugs through the biological membrane while reducing the dissolution of the mucosal cell membrane structure and components by Tween-20.

[0024] In some embodiments, the auxiliary material comprises glucose, sodium chloride and sodium sulfate.

[0025] In some embodiments, the mass ratio of glucose, sodium chloride and sodium sulfate is 1:(1.2-1.6):(0.2-0.4).

[0026] The second aspect of the present invention provides a method for preparing veterinary antiviral powder, comprising the following steps: mixing auxiliary materials, nano-drugs and compounding accelerators to obtain the veterinary antiviral powder.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides a kind of veterinary antiviral powder of adenosine, which improves the antiviral activity of adenosine in animals by making antiviral components into nano-drugs, reduces the dosage, and thus reduces the toxicity to the animals themselves; at the same time, the delivery and stability of nano-drugs in organisms are improved by compounding promoters.

[0029] 2. Among the nanomedicines provided by the present invention, seaweed polysaccharides and adenosine have antiviral activity. Through the principle of electrostatic adsorption, the negatively charged seaweed polysaccharides are adsorbed on the surface of the positively charged chitosan, and then polymerized with sodium tripolyphosphate to form nanomedicines, thereby improving the activity of seaweed polysaccharides in vivo and the encapsulation rate and drug loading of adenosine. DETAILED DESCRIPTION

[0030] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following examples are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Other combinations and various improvements within the concept of the present invention may be made without departing from the gist or scope of the present invention. It should be noted that the post-processing steps such as "centrifugation" and "freeze drying" that appear in the following preparation examples belong to routine operations of those skilled in the art and are selected according to actual operations.

[0031] In order to facilitate those skilled in the art to implement the present invention, some raw material manufacturers of the embodiments and comparative examples are described as follows:

[0032] Chitosan, seaweed polysaccharide, poloxamer 188, and Tween-20 can be purchased from any manufacturer;

[0033] Alkyl glycosides were purchased from Jiangsu Wanqi Biotechnology Co., Ltd. with model number APG0810.

[0034] Preparation Example 1

[0035] The preparation steps of nano drug A are as follows:

[0036] S1. Dissolve 2 g of chitosan in 1000 mL of 1.5 wt% acetic acid solution at room temperature, stir until there is no solid, adjust the pH to 5 with 10 wt% NaOH solution, add 0.1 g of adenosine, stir for 15 min, then add 1.4 g of EDC and 0.8 g of NHS and stir for 120 min. After stirring, add anhydrous ethanol until no precipitation occurs, centrifuge to obtain the supernatant, and freeze-dry to obtain a chitosan drug complex.

[0037] S2. Add 500 mL of 0.5 mg / mL seaweed polysaccharide solution to the chitosan drug complex obtained in step S1, stir at room temperature for 30 min, add 1000 mL of 0.2 mg / mL sodium tripolyphosphate solution, continue stirring for 30 min, add 15 g of poloxamer 188, continue stirring for 60 min, remove the precipitate by centrifugation, and freeze-dry to obtain nano drug A.

[0038] Preparation Example 2

[0039] The preparation steps of nano drug B are as follows:

[0040] The difference between this preparation example and preparation example 1 is that the amount of seaweed polysaccharide solution used in step S2 is 380 mL.

[0041] Preparation Example 3

[0042] The preparation steps of nano drug C are as follows:

[0043] The difference between this preparation example and preparation example 1 is that the amount of seaweed polysaccharide solution used in step S2 is 620 mL.

[0044] Preparation Example 4

[0045] The preparation steps of nano drug D are as follows:

[0046] The difference between this preparation example and preparation example 1 is that the concentration of the sodium tripolyphosphate solution in step S2 is 0.05 mg / mL.

[0047] Preparation Example 5

[0048] The preparation steps of nano drug E are as follows:

[0049] The difference between this preparation example and preparation example 1 is that the concentration of the sodium tripolyphosphate solution in step S2 is 0.35 mg / mL.

[0050] According to the guidelines for microparticle preparations in the 2020 edition of the Pharmacopoeia, the encapsulation efficiency and drug loading of the nanodrugs provided in the preparation example were determined, and the results are shown in Table 1.

[0051] Table 1 Encapsulation efficiency and drug loading of nanodrugs

[0052] Nanomedicine A B C D E Encapsulation rate% 85.8 80.8 82.7 80.3 83.1 Drug loading% 6.6 5.3 5.9 5.0 6.2

[0053] Example 1

[0054] A veterinary antiviral powder comprises the following raw materials, measured by weight: 23 parts of glucose, 33 parts of sodium chloride, 8 parts of sodium sulfate, 31 parts of nano drug A, 1.5 parts of Tween-20 and 3.5 parts of alkyl glycoside.

[0055] The preparation method of the veterinary antiviral powder in this embodiment is as follows: glucose, sodium chloride, sodium sulfate, nano drug A, Tween-20 and alkyl glycoside are uniformly mixed to obtain the veterinary antiviral powder.

[0056] Example 2

[0057] A veterinary antiviral powder comprises the following raw materials, measured by weight: 20 parts of glucose, 34 parts of sodium chloride, 6 parts of sodium sulfate, 28 parts of nano drug A, 1 part of Tween-20 and 3 parts of alkyl glycoside.

[0058] The preparation method of the veterinary antiviral powder in this embodiment is as follows: glucose, sodium chloride, sodium sulfate, nano drug A, Tween-20 and alkyl glycoside are uniformly mixed to obtain the veterinary antiviral powder.

[0059] Example 3

[0060] A veterinary antiviral powder comprises the following raw materials, measured by weight: 25 parts of glucose, 35 parts of sodium chloride, 8 parts of sodium sulfate, 34 parts of nano drug A, 2.1 parts of Tween-20 and 4.9 parts of alkyl glycoside.

[0061] The preparation method of the veterinary antiviral powder in this embodiment is as follows: glucose, sodium chloride, sodium sulfate, nano drug A, Tween-20 and alkyl glycoside are uniformly mixed to obtain the veterinary antiviral powder.

[0062] Example 4

[0063] This embodiment provides an antiviral drug powder for veterinary use and a preparation method thereof. The specific implementation method is the same as that of Example 1, except that the nano drug A in the raw material is replaced by an equal portion of nano drug B.

[0064] Example 5

[0065] This embodiment provides an antiviral drug powder for veterinary use and a preparation method thereof. The specific implementation method is the same as that of Example 1, except that the nano drug A in the raw material is replaced by an equal portion of nano drug C.

[0066] Example 6

[0067] This embodiment provides an antiviral powder for veterinary use and a preparation method thereof. The specific implementation method is the same as that of Example 1, except that the nano drug A in the raw material is replaced by an equal portion of nano drug D.

[0068] Example 7

[0069] This embodiment provides an antiviral drug powder for veterinary use and a preparation method thereof. The specific implementation method is the same as that of Example 1, except that the nano drug A in the raw material is replaced by an equal amount of nano drug E.

[0070] Example 8

[0071] This embodiment provides an antiviral powder for veterinary use and a preparation method thereof. The specific implementation method is the same as that of Example 1, except that Tween-20 in the raw material is replaced by an equal portion of alkyl glycoside.

[0072] Example 9

[0073] This embodiment provides an antiviral powder for veterinary use and a preparation method thereof. The specific implementation method is the same as that of Example 1, except that the alkyl glycoside in the raw material is replaced by an equal portion of Tween-20.

[0074] Comparative Example 1

[0075] This comparative example provides an antiviral powder for veterinary use. The difference between the raw materials and those of Example 1 is that adenosine is used instead of nano drug A, wherein the amount of adenosine used = 31 parts by weight of nano drug A × the drug loading of nano drug A (6.6%).

[0076] The specific preparation method of the veterinary antiviral powder in this comparative example is as follows: Glucose, sodium chloride, sodium sulfate, adenosine arabinoside, Tween-20 and alkyl glycoside are uniformly mixed to obtain the veterinary antiviral powder.

[0077] Performance Testing

[0078] Establishment of in vivo infection model of porcine pseudorabies virus (PRV) and grouping:

[0079] Twenty 10-week-old piglets were randomly divided into 10 groups, with 2 piglets in each group, and injected with 1 mL of 10 4 TCID 50 (half tissue culture infectious dose) of virus liquid, strictly isolated and raised for 3 consecutive weeks to obtain infected piglets.

[0080] 1. Stability test

[0081] Ten groups of infected piglets corresponding to Examples 1 to 9 and Comparative Example 1 were fed with the same mass of antiviral powder, and the intestinal contents were extracted 1 hour after feeding. The same mass of intestinal contents were placed on culture dishes of the same size, and the morphology of the nanomedicines therein was observed to see if they were disintegrated. The stability was determined as follows: 0-10% disintegration, excellent stability; 10-30% disintegration, good stability; 30-50% disintegration, general stability; ≥50%, poor stability.

[0082] 2. Antiviral test

[0083] The plasma of the infected piglets was collected and the plasma viral load T0 was determined. The 10 groups of infected piglets corresponding to Examples 1 to 9 and Comparative Example 1 were fed with the same mass of antiviral powder. The plasma after drug administration was collected 4 hours after feeding, and the plasma viral load T1 was determined. The reduction rate of the viral load in the plasma was calculated, and the formula was [1-(T1 / T0)]×100%.

[0084] The above test results are shown in Table 1.

[0085] Table 1 Performance test results

[0086] stability Viral load reduction rate (%) Example 1 excellent 87.3 Example 2 excellent 86.1 Example 3 excellent 86.8 Example 4 good 75.4 Example 5 good 73.2 Example 6 good 76.9 Example 7 good 78.0 Example 8 Difference 79.5 Example 9 generally 80.7 Comparative Example 1 good 74.3

[0087] It can be seen from the data in Table 1 that the nano drugs in the veterinary antiviral powders of Examples 1-3 have high stability and antiviral activity in the infected piglets; compared with Example 1, the nano drugs in the antiviral powders of Examples 4-5 changed the dosage of seaweed polysaccharide solution during preparation, and Examples 6-7 changed the concentration of sodium tripolyphosphate solution. It can be seen from the data in Table 1 that these changes in preparation process are not conducive to the encapsulation and drug loading of nano drugs, and combined with the viral load reduction rate in Table 1, it can be seen that these changes will also reduce the stability and antiviral activity of the drug. Compared with Example 1, Examples 8-9 use a single promoter, which is not conducive to maintaining the stability of nano drugs in infected piglets, and also has a certain inhibition on the antiviral effect. Compared with Example 1, Comparative Example 1 directly uses adenosine. At the same dosage, making adenosine into nano drugs significantly improves its antiviral activity.

[0088] The embodiments and comparative examples described above do not impose any form of limitation on the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. An antiviral powder for veterinary use, characterized in that: The invention comprises the following raw materials by weight: 60 to 68 parts of auxiliary materials, 28 to 34 parts of nano drugs and 4 to 7 parts of compounding promoters; wherein the encapsulation rate of the nano drugs is ≥80.0%, and the drug loading is ≥5.0%.

2. The veterinary antiviral powder according to claim 1, characterized in that: The preparation steps of the nano drug are as follows: S1. Dissolve chitosan in acetic acid solution at room temperature, stir until there is no solid, adjust the pH to 4.6-5.4, add adenosine, stir for 10-15 minutes, then add EDC and NHS and stir for 100-150 minutes, add anhydrous ethanol after stirring until no precipitation, centrifuge to obtain the supernatant, and freeze-dry to obtain a chitosan drug complex; S2, adding the seaweed polysaccharide solution to the chitosan drug complex obtained in step S1, stirring at room temperature for 25 to 35 minutes, adding the sodium tripolyphosphate solution, continuing to stir for 25 to 35 minutes, adding poloxamer 188, continuing to stir for 50 to 70 minutes, centrifuging to remove the precipitate, and freeze-drying to obtain the nano drug.

3. The veterinary antiviral powder according to claim 2, characterized in that: In step S1, the mass ratio of chitosan to adenosine is 1:(0.03-0.07).

4. The veterinary antiviral powder according to claim 2, characterized in that: In step S2, the concentration of the seaweed polysaccharide solution is 0.3-0.8 mg / mL, and the concentration of the sodium tripolyphosphate solution is 0.1-0.3 mg / mL.

5. The veterinary antiviral powder according to claim 4, characterized in that: The ratio of the chitosan in step S1 to the seaweed polysaccharide solution in step S2 is 1 mg: (0.2-0.3) mL.

6. The veterinary antiviral powder according to claim 1, characterized in that: The compounding accelerator comprises Tween-20 and alkyl glycoside.

7. The veterinary antiviral powder according to claim 6, characterized in that: The mass ratio of Tween-20 to alkyl glycoside is (0.33-0.45):

1.

8. The veterinary antiviral powder according to claim 1, characterized in that: The auxiliary materials include glucose, sodium chloride and sodium sulfate.

9. The veterinary antiviral powder according to claim 8, characterized in that: The mass ratio of the glucose, sodium chloride and sodium sulfate is 1:(1.2-1.6):(0.2-0.4).

10. A method for preparing the veterinary antiviral powder according to any one of claims 1 to 9, characterized in that: The method specifically comprises the following steps: mixing auxiliary materials, nano-drugs and compounding accelerators to obtain the veterinary antiviral powder.