CN114404505B

CN114404505B - Traditional Chinese medicine preparation mainly prepared from south medicines and used for preventing and controlling African swine fever and preparation method

Info

Publication number: CN114404505B
Application number: CN202210180475.8A
Authority: CN
Inventors: 雍燕; 颜振炽; 方炳虎; 刘国昌
Original assignee: Guangdong Wens Dahuanong Biotechnology Co ltd
Current assignee: Guangdong Wens Dahuanong Biotechnology Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2023-01-17
Anticipated expiration: 2042-02-25
Also published as: CN114404505A

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine preparation mainly prepared from south China medicines and used for preventing and controlling African swine fever and a preparation method thereof. The traditional Chinese medicine preparation mainly prepared from south Chinese medicines comprises the following components in parts by weight: 5 to 8 parts of areca, 3 to 7 parts of cassia twig, 3 to 7 parts of beautiful millettia root, 3 to 7 parts of coptis root, 3 to 7 parts of baical skullcap root, 3 to 5 parts of heartleaf houttuynia herb, 3 to 5 parts of south indigowoad root, 3 to 5 parts of echinacea and 1 to 2 parts of liquoric root. The traditional Chinese medicine preparation provided by the invention is mainly prepared from south Chinese herbs, is a pure traditional Chinese medicine preparation, is natural and safe, has low toxic and side effects, is synergistic in components, can effectively inhibit the replication of the African swine fever virus in vitro, improves the immunity of pigs, enhances the resistance to the viruses such as the African swine fever and the like, reduces the morbidity of the diseases, and assists the development of the swine industry under the condition of normalization of the African swine fever.

Description

Traditional Chinese medicine preparation mainly prepared from south medicines and used for preventing and controlling African swine fever and preparation method

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine preparation mainly prepared from south China medicines and used for preventing and controlling African swine fever and a preparation method thereof.

Background

African swine fever is an acute, virulent and highly pathogenic infectious disease caused by African Swine Fever Virus (ASFV), which is classified as a legal report animal epidemic disease by the world animal health Organization (OIE), and is classified as a type of animal epidemic disease in China. In 1921, ASF was first discovered in kenya and then spread to over 60 countries in west africa, europe, asia, causing severe losses to agriculture and animal husbandry worldwide. In 2018, in 8 and 1 month, after the Shenyang of Liaoning in China discovers the first African swine fever, the epidemic situation of the African swine fever rapidly spreads to many places in China due to the live pig space circulation mode of 'east transport of west pigs and south transport of north pigs', so that a huge threat is formed to the live pig breeding industry in China.

The traditional Chinese medicine is unique in medical science in China, has a long history, and makes important contribution to medical care of the masses for thousands of years. The traditional Chinese medicine antivirus has become the hotspot of modern medicine and pharmacology research, and modern research shows that part of traditional Chinese medicines have the effect of inhibiting and killing viruses. Under the condition that the modern medicine for preventing and controlling the African swine fever is lack of effective products, the method for preventing and controlling the African swine fever by combining the traditional Chinese medicine and the modern preparation technology has obvious advantages. However, the distribution of medicinal material resources has obvious regional characteristics, and the cost control requirement of the veterinary traditional Chinese medicine is higher, so that the medicinal materials in a specific region are produced and used nearby, and the development trend of the veterinary traditional Chinese medicine is better met. The south medicine is a traditional Chinese medicine produced in south of Yangtze river and north of Lingnan, has rich resources, and multiple researches show that the south medicines such as cinnamon, areca nut, polygonum hydropiper, houttuynia cordata, common andrographis herb, wrinkled gianthyssop herb, south isatis root and the like have the effects of sterilizing, resisting inflammation, detoxifying, enhancing immunity and the like, and have great development and application values.

Currently, there is no effective vaccine and drug for african swine fever. Strict biological control and raising of swinery immunity are the most important control measures at present. The invention combines the traditional Chinese medicine theory, explores the traditional Chinese medicine pathogenesis of the ASF, researches and develops the traditional Chinese medicine preparation for preventing and controlling the ASF and the application method thereof through in-vitro and in-vivo tests, provides a new means for preventing and controlling the ASF, and provides a solid guarantee for the breeding of live pigs.

Disclosure of Invention

Aiming at the defects generally existing in the prior art, the invention provides a traditional Chinese medicine preparation mainly prepared from south China medicines and used for preventing and controlling African swine fever and a preparation method thereof. The traditional Chinese medicine preparation provided by the invention is a pure traditional Chinese medicine preparation mainly comprising south medicines, is natural and safe, has low toxic and side effects, can effectively inhibit the replication of African swine fever viruses in vitro, improves the immunity of pigs, enhances the resistance to the African swine fever viruses and the like, and reduces the morbidity of diseases.

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

a traditional Chinese medicine preparation comprises the following components in parts by weight: 5-8 parts of areca, 3-7 parts of cassia twig, 3-7 parts of beautiful millettia root, 3-7 parts of coptis root, 3-7 parts of baical skullcap root, 3-5 parts of heartleaf houttuynia herb, 3-5 parts of south indigowoad root, 3-5 parts of echinacea and 1-2 parts of liquorice.

Preferably, the composition consists of the following components in parts by weight: 7 parts of areca-nut, 5 parts of cassia twig, 5 parts of beautiful millettia root, 5 parts of coptis root, 5 parts of baical skullcap root, 4 parts of heartleaf houttuynia herb, 4 parts of south indigowoad root, 4 parts of echinacea and 1.5 parts of liquorice.

The invention also provides a preparation method of the traditional Chinese medicine preparation, which comprises the following steps: respectively weighing the areca, the cassia twig, the beautiful millettia root, the coptis root, the baical skullcap root, the heartleaf houttuynia herb, the south indigowoad root, the purple daisy and the liquoric root in parts by weight, crushing by using a high-efficiency universal crusher, sieving by using a 40-120-mesh sieve, and then mixing and stirring uniformly to obtain the traditional Chinese medicine.

Preferably, the mixing and stirring conditions are 500-800 rpm for 10-20 min.

The invention also provides application of the traditional Chinese medicine preparation in preparation of African swine fever feed.

Preferably, the addition amount of the traditional Chinese medicine preparation and the feed is 6-10 g/kg.

In the experimental process, according to clinical dialectics (wei-qi-ying-blood dialectics) carried out in 2020, the prevention and treatment principle is determined as follows: the preparation method comprises the steps of tonifying qi and spleen, promoting qi circulation and descending adverse qi, clearing away heat and toxic materials, cooling blood and removing speckles, and is characterized in that a plurality of different formulas are arranged, samples are prepared, and the application evaluation is respectively carried out in pig farms such as Guangxi province and Hunan province. According to the application conditions of different areas, analysis is carried out, the correctness of the basic direction is determined, the formula is gradually perfected by combining the defects in the application, the preparation process and the quality standard (internal control) are determined through sample trial production and quality research, and finally the traditional Chinese medicine formula is successfully obtained.

In the process of preparing the feed for preventing the African swine fever to feed pigs, the feed can adopt the following feeding mode: (1) The medicinal materials in the formula are crushed into powder of 40 meshes to 120 meshes by a high-efficiency crusher, and the powder is mixed uniformly to prepare the traditional Chinese medicine powder. Adding the traditional Chinese medicine powder into feed in a dosage of 6-10 g per kilogram of feed, stirring and mixing uniformly, and feeding; (2) The medicinal materials in the formula are crushed into powder of 40 meshes to 120 meshes by a high-efficiency crusher, and the powder is mixed uniformly to prepare the traditional Chinese medicine powder. The traditional Chinese medicine powder is fermented by using a microecological preparation for use, and the specific method comprises the following steps: 6.5kg of traditional Chinese medicine powder, 5kg of microecological preparation prepared from saccharomycetes, bacillus subtilis, lactic acid bacteria and the like, 5kg of brown sugar and 100kg of pig feed are added into 1 ton of clean water (35-40 ℃). The materials are well mixed and put into a fermentation barrel to be sealed by a film, the film is tightly bound by a rubber band, and the fermentation is carried out for 24 to 72 hours (24 to 48 hours in summer in high temperature weather and 48 to 72 hours in winter and spring at the environmental temperature of about 28 ℃). The unsealed fermentation water can smell a strong acid fragrance after the fermentation is finished, and the pH value is detected to be below 3.8. Directly feeding the fermented feed containing the medicine; (3) The medicinal materials in the formula are crushed into 10-20 meshes of coarse powder, water which is 12 times of the total weight of the medicinal materials is added, the mixture is decocted for 1 hour, liquid medicine is filtered, the liquid medicine is further concentrated into thick paste, and the thick paste, dregs of a decoction and feed are uniformly mixed and fed. The feeding mode is selected according to the scale, season, feeding habit and the like of a pig farm.

Compared with the prior art, the traditional Chinese medicine preparation provided by the invention has the following advantages:

(1) The invention is a pure Chinese medicinal preparation, is natural and safe, has low toxic and side effects, has synergistic components, can effectively inhibit the replication of the African swine fever virus in vitro, improves the immunity of pigs, enhances the resistance to the African swine fever virus and the like, reduces the morbidity of diseases, and assists the development of the swine industry under the condition of normalization of the African swine fever.

(2) The invention selects south medicinal materials with wide sources for composing the prescription, has unique regional advantages, is more suitable for the practical application of veterinary medicaments in livestock breeding, provides a plurality of application schemes for preventing and controlling the African swine fever of the prescription, and can be suitable for preventing and controlling diseases in different-scale farms, different medication habits and different seasons.

Drawings

FIG. 1 is a graph showing the results of in vitro virus inhibition experiments;

FIG. 2 is a graph showing the results of an indirect immunofluorescence assay;

FIG. 3 is a graph showing the results of a virus adsorption test;

FIG. 4 is a graph showing the results of a virus penetration test;

FIG. 5 is a graph showing the results of the virus replication assay.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The specific techniques or conditions not mentioned in this example were followed by procedures and apparatus instructions in accordance with conventional techniques in the art; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1A Chinese medicinal preparation

The traditional Chinese medicine preparation comprises the following components in parts by weight: 5 parts of areca nut, 3 parts of cassia twig, 3 parts of beautiful millettia root, 3 parts of coptis root, 3 parts of baical skullcap root, 3 parts of heartleaf houttuynia herb, 3 parts of south indigowoad root, 3 parts of echinacea and 1 part of liquorice.

The preparation process of the traditional Chinese medicine preparation is as follows: respectively weighing the areca, the cassia twig, the beautiful millettia root, the coptis root, the baical skullcap root, the heartleaf houttuynia herb, the south indigowoad root, the purple daisy and the liquoric root in parts by weight, crushing by using a high-efficiency universal crusher, sieving by using a 40-mesh sieve, stirring for 20min at 500rpm, and mixing and stirring uniformly to obtain the traditional Chinese medicine.

Example 2A Chinese medicinal preparation

The traditional Chinese medicine preparation comprises the following components in parts by weight: 8 parts of areca-nut, 7 parts of cassia twig, 7 parts of beautiful millettia root, 7 parts of coptis root, 7 parts of baical skullcap root, 5 parts of heartleaf houttuynia herb, 5 parts of south indigowoad root, 5 parts of echinacea and 2 parts of liquorice.

The preparation process of the traditional Chinese medicine preparation is as follows: respectively weighing the areca, the cassia twig, the beautiful millettia root, the coptis root, the baical skullcap root, the heartleaf houttuynia herb, the south indigowoad root, the purple daisy and the liquoric root in parts by weight, crushing by using a high-efficiency universal crusher, sieving by using a 120-mesh sieve, stirring for 10min at 800rpm, and mixing and stirring uniformly to obtain the traditional Chinese medicine.

Example 3A Chinese medicinal preparation

The traditional Chinese medicine preparation comprises the following components in parts by weight: 7 parts of areca-nut, 5 parts of cassia twig, 5 parts of beautiful millettia root, 5 parts of coptis root, 5 parts of baical skullcap root, 4 parts of heartleaf houttuynia herb, 4 parts of south indigowoad root, 4 parts of echinacea and 1.5 parts of liquorice.

The preparation process of the traditional Chinese medicine preparation is as follows: respectively weighing the areca, the cassia twig, the beautiful millettia root, the coptis root, the baical skullcap root, the heartleaf houttuynia herb, the south isatis root, the purple daisy and the liquorice in parts by weight, crushing by using a high-efficiency universal crusher, sieving by using a 80-mesh sieve, and stirring at 600rpm for 15min to mix and stir uniformly to obtain the traditional Chinese medicine.

Test example 1 in vitro virus inhibition test

1. Test materials: the test cells of the chinese herbal preparation prepared in example 3 were primary Porcine Alveolar Macrophages (PAMs), african swine fever virus strain: ASFV/China/GZ201801 separated and identified by national African swine fever regional laboratory (Guangzhou), and the virus concentration is 10 ⁶ HAD ₅₀ /mL；

2. The test method comprises the following steps:

2.1 preliminary experiments section: PAMs were plated in 96-well plates, and 1 ₂ The incubator is used for 48 hours. CCK8 was measured after 2 days of culture.

2.2 official test: in order to avoid the influence of the disinfectant on the cell growth, the formal test is adjusted according to the result of the preliminary test, namely the disinfectant reacts with the virus suspension, then the disinfectant is diluted to a safe concentration, and then the disinfectant is applied to the cell for incubation.

2.2.1 Indirect immunofluorescence assay

Inoculating PAM cells into 48-well plate, allowing cells to adhere, inoculating cells with 100-fold diluted ASFV virus solution at 250. Mu.L/well, at 37 ℃ and 5% CO ₂ After 2h of incubator culture, the Chinese medicinal preparation was diluted to 1 ₂ After culturing in an incubator for 48h, performing an indirect immunofluorescence test.

2.2.2 Virus adsorption experiments

Inoculating PAM cell in 24-well plate, allowing cell to adhere to wall, discarding culture solution, and diluting with 100 times of ASFV virus solution the traditional Chinese medicine preparation was diluted to 1 ₂ And collecting samples after the cultivation in an incubator for 48 hours, and performing QPCR (quantitative PCR) test.

2.2.3 Virus penetration test

The PAM cells were seeded in a 24-well plate, the culture solution was discarded after the cells were attached to the wall, the cells were seeded with 100-fold diluted ASFV virus solution, 500 μ L/well, and after incubation for 1h in a refrigerator at 4 ℃, the cells were washed 3 times with serum-free RPMI Medium1640 Medium, the test compound was diluted to 1 ₂ Culturing in culture box for 1h, washing with serum-free RPMI Medium1640 Medium for 3 times, adding 10% FBS-containing RPMI Medium1640 Medium again, and making 5% CO at 37 deg.C ₂ And collecting samples after the cultivation in an incubator for 48 hours, and performing QPCR (quantitative PCR) test.

2.2.4 Virus replication assay

Inoculating PAM cells into 24-well plate, allowing cells to adhere, discarding culture solution, inoculating cells with 50-fold diluted ASFV virus solution, 500 μ L/well, at 37 deg.C, 5% ₂ Incubate for 2h, dilute the test compound to 1, 500, 1, 1000, 1 ₂ After 48h incubation in the incubator, virus supernatants were collected for HAD50 determination and QPCR assay.

3. The experimental results are as follows: as shown in FIG. 1, the preliminary experiment results show that the Chinese medicine has no toxicity to PAMs when diluted 500 times, and the CC50 value is 0.01741 (when diluted 57 times approximately), i.e. the concentration of the medicine with CC50 of 0.01741 is approximately 57 times the concentration of the medicine, which has little influence; the indirect immunofluorescence test result is shown in figure 2, and the figure shows that the traditional Chinese medicine inhibits the replication of viruses in a dose-dependent manner, and the traditional Chinese medicine can inhibit the replication of more than 90 percent of viruses when diluted by 1000 times; the virus adsorption test is shown in figure 3, and the results show that the traditional Chinese medicine can inhibit the adsorption of ASFV under the dilution of 1; the results of the virus penetration test are shown in fig. 4, and the results show that the traditional Chinese medicine can inhibit the internalization of ASFV at the dilution of 1.

From the results, the traditional Chinese medicine preparation provided by the invention inhibits the proliferation of ASFV in PAMs cells in a dose-dependent relationship, has an EC50 value of 0.0001195 and a dilution factor of about 8368, has no toxic effect on the PAMs cells when diluted 500 times, has a CC50 of 0.01741 and a dilution factor of about 57, and has a selection index (SI = CC50/EC 50) of 145, and can block the adsorption and internalization stages of ASFV on the PAMs cells.

Test example 2 African Swine fever control test

1. Test samples: the Chinese medicinal preparation prepared in examples 1-3;

2. test subjects: 120 pork pigs of 100 days old are randomly divided into 4 groups, and each group has 30 pigs; the control group was given no drug, and the test groups were given the chinese herbal preparations of the present invention in the examples 1 to 3 groups, respectively.

3. The test method comprises the following steps: the traditional Chinese medicine preparation prepared in the embodiment 1-3 is respectively decocted for 10-15 minutes by adding water at the dose of 20g per pig per day in three consecutive months from 12 months 1 day in 2020 to 2 months and 28 days in 2021, and is cooled and mixed with the soup and the slag for use, and the used feed is a wenshi pork pig compound feed (consisting of corn, bean pulp, fish meal and the like, and does not contain any chemical medicine or traditional Chinese medicine by detection). The health status and mortality of each group of pigs were observed and recorded daily, and blood was collected at the middle of the test for african swine fever antibody detection.

4. And (3) test results: the specific test results are shown in tables 1 and 2.

Table 1-1 clinical symptom score table for pigs of examples 1-2 groups

TABLE 1-2 TABLE OF RECORDING OF THE clinicopathic symptomatology OF PIGS IN EXAMPLE 3 AND CONTROL GROUP

TABLE 2 African swine fever antibody test results after 2 months of feeding

Test sample	X％	Determination
			EXAMPLE 1 group	102.6	+
EXAMPLE 2 group	102.7	+
			EXAMPLE 3 group	102.9	+
Control group	96.6	+

And (3) judging standard: x% is more than or equal to 50% as positive, X% is more than or equal to 40% as negative, and suspicious results are obtained between the positive and the negative

After 3 months of test, the swinery fed with the traditional Chinese medicine preparation prepared by the embodiment of the invention has no obvious symptom, the growth of the animal population is good, and the weight gain of the animals is relatively average; in contrast, 15 pigs in the control group died with a mortality rate of 25%, and the surviving pigs had large differences in growth status and large weight deviations. The pigs in the test group are superior to the control group in feed intake, mental state, clinical symptoms, mortality, weight gain and the like. From the detection result of the African swine fever antibody, after the African swine fever antibody is fed by the traditional Chinese medicine preparation for 2 months, the level of the African swine fever antibody in a test group is higher, and the level of the antibody between pigs is not obviously different, so that the traditional Chinese medicine preparation has the effects of enhancing the immunity, improving the resistance to viruses and being beneficial to the generation of the antibody.

Test example 3 clinical verification test

1. Test samples: the Chinese medicinal preparation of example 3;

2. the test process comprises the following steps: the traditional Chinese medicine preparation is directly added into the feed for use at a dosage of 10g/kg feed in a plurality of pig farms in Xiaochang pig farms in Hubei province, and is continuously used for 8-15 days to observe the overall state of a pig herd and count the death and culling rate.

3. And (3) test results: specific test results are shown in table 3.

As can be seen from the above Table 3, during the use period, the mortality of the swinery is obviously lower than that before the use, and half of the farmer pigs have mortality within 1%, and the overall state of the swinery is obviously improved after the farmer reaction is used; this demonstrates precisely the important role of the Chinese medicinal preparation of the present invention.

It should be noted that although the above embodiments have been described, once the basic inventive concept is obtained, other variations and modifications can be made to these embodiments by those skilled in the art, so that the above embodiments are only examples of the present invention, and not to limit the scope of the present invention, and all the modifications made by the equivalent structures or equivalent processes in the present specification, or directly or indirectly applied to other related technical fields are included in the scope of the present invention.

Claims

1. The traditional Chinese medicine preparation for preventing and controlling African swine fever is characterized by being prepared from the following components in parts by weight: 5 to 8 parts of areca, 3 to 7 parts of cassia twig, 3 to 7 parts of beautiful millettia root, 3 to 7 parts of coptis root, 3 to 7 parts of baical skullcap root, 3 to 5 parts of heartleaf houttuynia herb, 3 to 5 parts of south indigowoad root, 3 to 5 parts of echinacea and 1 to 2 parts of liquoric root.

2. The traditional Chinese medicine preparation as claimed in claim 1, which is prepared from the following components in parts by weight: 7 parts of areca-nut, 5 parts of cassia twig, 5 parts of beautiful millettia root, 5 parts of coptis root, 5 parts of baical skullcap root, 4 parts of heartleaf houttuynia herb, 4 parts of south isatis root, 4 parts of echinacea and 1.5 parts of liquorice.

3. A method for preparing the traditional Chinese medicine preparation of claim 1, which is characterized by comprising the following steps: respectively weighing the areca, the cassia twig, the beautiful millettia root, the coptis root, the baical skullcap root, the heartleaf houttuynia herb, the south indigowoad root, the purple daisy and the liquoric root in parts by weight, crushing by using a high-efficiency universal crusher, sieving by using a 40-120-mesh sieve, and then mixing and stirring uniformly to obtain the traditional Chinese medicine.

4. The method according to claim 3, wherein the mixing and stirring are carried out at 500 to 800rpm for 10 to 20min.

5. The application of the traditional Chinese medicine preparation of claim 1 in preparing an African swine fever prevention and control feed.

6. The use of claim 5, wherein the amount of the Chinese medicinal preparation added to the feed is 6-10 g/kg.