Composition for resisting fish herpes virus, compound Chinese herbal medicine preparation, preparation method and application

Abstract

The invention relates to the technical field of aquaculture, in particular to an anti-fish herpesvirus composition, a compound Chinese herbal medicine preparation, a preparation method and application. The composition for resisting herpes viruses of fishes comprises the following components in percentage by weight: 5-10% of a radix salviae miltiorrhizae extract, 10-30% of a herba taraxaci extract, 20-40% of a radix angelicae dahuricae extract and 20-65% of a szechwan chinaberry fruit extract. The inhibition effect of the composition on herpes viruses is superior to that of antibiotics. The compound Chinese herbal medicine preparation for resisting the fish herpes virus comprises 30-90% of a composition C (prepared by mixing a composition A and a composition B prepared from the composition for resisting the fish herpes virus) and 10-70% of a composition D. The compound Chinese herbal medicine preparation can inhibit the division function of the herpes virus, so that the herpes virus loses the reproductive capacity, and the curative effect of the herpes virus is improved. The composition has the advantages of quick action, no drug resistance, no side effect and no environmental pollution.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and specifically relates to a composition, a compound Chinese herbal medicine preparation, a preparation method and an application for resisting fish herpes virus. Background Art

[0002] Fish is a major source of protein for people. However, with the deterioration of the aquaculture environment and the change of aquaculture patterns, fish diseases have become one of the main reasons hindering the growth of the fishery economy. Herpesviruses are a class of enveloped double-stranded DNA viruses that can infect a variety of vertebrates including fish. Fish herpesviruses are important pathogens in the aquaculture industry, capable of causing a variety of serious diseases and causing significant losses to the global fishery economy. These viruses have host specificity, and different herpesvirus strains usually only infect specific fish species.

[0003] Existing studies have developed vaccines against fish herpesviruses. These vaccines stimulate the fish immune system to produce specific antibodies by encoding viral antigen genes, thereby improving the disease resistance of fish. However, vaccination with herpesvirus vaccines may cause stress reactions in fish, and currently, the variety of commercial vaccines is limited, unable to comprehensively cover the herpesvirus diseases of major aquaculture species, and a complete comprehensive prevention and control system for disease immunity cannot be formed.

[0004] Chinese herbal medicines are widely used in aquaculture to prevent and treat fish herpesvirus infections. Chinese herbal medicines contain a variety of natural active ingredients, such as flavonoids, polysaccharides, organic acids and saponins, etc. These ingredients not only have a direct antiviral effect, but also can regulate the immune function of fish. Research shows that certain Chinese herbal medicine extracts can exert antiviral effects by interfering with links such as herpesvirus adsorption, invasion or replication. Compared with chemical drugs, Chinese herbal medicines are not likely to cause drug resistance in viruses and are more environmentally friendly.

[0005] However, the treatment of herpesvirus infections with Chinese herbal medicines also has certain limitations. Its treatment effect usually takes a long time to appear, and it may be difficult to quickly control the condition in case of acute infections; in addition, due to the diverse sources and complex components of Chinese herbal medicines, quality control is relatively difficult, which may affect the stability and repeatability of the treatment effect. At present, researchers are working hard to improve the efficacy of Chinese herbal medicines against herpesviruses by extracting and purifying effective components, optimizing the administration plan and other means. Summary of the Invention

[0006] In view of the technical problems of the deficiencies and defects of the existing herpesvirus treatment methods, the present invention provides a composition, a compound Chinese herbal medicine preparation, a preparation method and an application for resisting fish herpes virus.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a composition against fish herpesvirus, comprising components in the following weight percentages: 5-10% of Salvia miltiorrhiza extract, 10-30% of Taraxacum mongolicum extract, 20-40% of Angelica dahurica extract, and 20-65% of Melia toosendan extract.

[0009] In the present invention, the preparation method of the Salvia miltiorrhiza extract, Taraxacum mongolicum extract, Angelica dahurica extract or Melia toosendan extract is as follows: Mix the powder of Salvia miltiorrhiza, Taraxacum mongolicum, Angelica dahurica or Melia toosendan with ethanol at a mass-to-volume ratio of 1:9 - 5:5 g / cm 3 Extract, concentrate and dry to obtain the extract.

[0010] In one embodiment, the drying method includes spray drying, fluidized bed drying, freeze drying or rotary flash drying.

[0011] In one embodiment, the drying temperature is 50 - 70 °C and the drying time is 1 - 4 hours.

[0012] The present invention also provides a compound Chinese herbal medicine preparation against fish herpesvirus, comprising components in the following weight percentages: 30 - 90% of composition C and 10 - 70% of composition D; based on the mass of composition C being 100%, composition C is composed of 30 - 60% of composition A and 40 - 70% of composition B; based on the mass of composition A being 100%, composition A is composed of 5 - 15% of Astragalus membranaceus powder, 10 - 20% of Scutellaria baicalensis powder, 10 - 30% of Glycyrrhiza glabra powder and 45 - 75% of corn starch; composition B is obtained by dissolving the composition against fish herpesvirus in ethanol and then drying; based on the mass of composition D being 100%, composition D is composed of 20 - 60% of Astragalus membranaceus extract and 40 - 80% of Scutellaria baicalensis extract. In one embodiment, the compound Chinese herbal medicine preparation is composed of 60 - 90% of composition C and 10 - 40% of composition D. In one embodiment, composition D is composed of 20 - 40% of Astragalus membranaceus extract and 60 - 80% of Scutellaria baicalensis extract.

[0013] In the present invention, the preparation methods of the Astragalus membranaceus extract and Scutellaria baicalensis extract are as follows: Select Astragalus membranaceus or Scutellaria baicalensis as the raw material, powder it, and mix the powder with ethanol at a mass-to-volume ratio of 1:9 - 5:5 g / cm 3 Extract, concentrate and dry to obtain the extract.

[0014] The present invention also provides a preparation method of the compound Chinese herbal medicine preparation against fish herpesvirus, which is obtained by mixing composition A and composition B to obtain composition C, and then mixing composition C and composition D.

[0015] The present invention also provides an application of the composition or the compound Chinese herbal medicine preparation in the preparation of drugs for preventing and / or treating fish herpesvirus diseases.

[0016] Advantages of the present invention over the prior art:

[0017] The compound Chinese herbal medicine preparation for resisting fish herpes virus of the present invention contains four active ingredients: dihydrochalcone, chalcone, steroidal alkaloid, and umbelliferone. When the content of dihydrochalcone is in the range of 15 - 50 μg / ml, it can effectively improve the anti-inflammatory ability of grass carp, crucian carp, common carp, mandarin fish, sea bass, and grouper against herpes virus, inhibit the splitting function of herpes virus, make herpes virus lose its reproductive ability, promote fish metabolism and non-specific immunity, and has a fast action, no drug resistance, no side effects, and does not pollute the environment. Among them, the compound Chinese herbal medicine preparation of the present invention can increase the survival rate of grouper raised in a serious herpes virus disease environment to 95%. Detailed implementation manners

[0018] The present invention provides a composition for resisting fish herpes virus, comprising the following components in weight percentages: 5 - 10% of salvia miltiorrhiza extract, 10 - 30% of dandelion extract, 20 - 40% of angelica dahurica extract, and 20 - 65% of toosendan fruit extract.

[0019] In the present invention, the fish includes any one or several of grass carp, crucian carp, common carp, mandarin fish, sea bass, and grouper.

[0020] In the present invention, the preparation methods of the salvia miltiorrhiza extract, dandelion extract, angelica dahurica extract, or toosendan fruit extract are as follows: Select salvia miltiorrhiza, dandelion, angelica dahurica, or toosendan fruit as raw materials, powder them, and extract the powder with ethanol according to a mass-volume ratio of 1:9 - 5:5 g / cm 3 Then concentrate and dry to obtain. The present invention has no special limitation on the specific sources of salvia miltiorrhiza, dandelion, angelica dahurica, and toosendan fruit, and commercially available products can be used.

[0021] In the present invention, the raw materials of salvia miltiorrhiza, dandelion, angelica dahurica, or toosendan fruit are dried first and then powdered; as an implementation manner, the drying temperature is 80 - 100°C and the drying time is 1 - 2 hours, which is beneficial for subsequent pulverization into uniform fine powder. As an implementation manner, after powdering, it is sieved through a sieve with a mesh size of less than 90 meshes, and further through a 200-mesh sieve to promote the dissolution of active substances in the raw materials during ethanol extraction. In the present invention, the powder is extracted with ethanol according to a mass-volume ratio of 1:9 - 5:5 g / cm 3 If not otherwise specified, the ethanol in the present invention is ethanol with a volume concentration of 75 - 85%. As an implementation manner, the mass-volume ratio of the powder to ethanol can be optionally 1:9, 2:8, 3:7, 4:6, and 5:5 g / cm 3. As an implementation method, the time for ethanol extraction is 20 - 40 minutes, and further preferably 25 - 35 minutes. After the ethanol extraction of the present invention, suction filtration is carried out to remove insoluble impurities, and the solution after suction filtration is concentrated and dried. The concentration is preferably vacuum concentration; as an implementation method, the pressure of the vacuum concentration is 80 - 100Kp, the temperature is 30 - 50°C, and it is concentrated to 1 / 5 - 1 / 8 of the original volume ， to obtain a concentrated solution. The drying methods of the concentrated solution include spray drying, fluidized bed drying, freeze drying or rotary flash drying; as an implementation method, the drying temperature is 50 - 70°C, and the drying time is 1 - 4 hours; as another implementation method, the drying temperature is 55 - 65°C, and the drying time is 2 - 3 hours. After drying is completed, the extracts of each Chinese medicinal material are obtained.

[0022] The present invention also provides a compound Chinese herbal medicine preparation for anti - fish herpes virus, which comprises the following components in weight percentages: 30 - 90% of composition C and 10 - 70% of composition D; as an implementation method, it comprises the following components in weight percentages: 40 - 80% of composition C and 20 - 60% of composition D. Mixing composition C and composition D in proportion gives the compound Chinese herbal medicine preparation.

[0023] In the present invention, based on the mass of composition C being 100%, composition C is composed of 30 - 60% of composition A and 40 - 70% of composition B. As an implementation method, composition C is composed of 35 - 55% of composition A and 45 - 65% of composition B. Mixing composition A and composition B in proportion gives composition C.

[0024] In the present invention, based on the mass of composition A being 100%, composition A is composed of 5 - 15% of astragalus powder, 10 - 20% of scutellaria powder, 10 - 30% of licorice powder and 45 - 75% of corn starch. In the present invention, the preparation methods of astragalus powder, scutellaria powder or licorice powder in composition A include selecting astragalus, scutellaria and licorice as raw materials, drying and powdering. As an implementation method, the drying temperature is 80 - 100°C, and the drying time is 1 - 2 hours; after powdering, it is sieved through a sieve with a mesh size below 90 meshes. Mixing the above components in proportion gives composition A. The present invention has no special limitation on the specific sources of astragalus, scutellaria, licorice and corn starch, and commercially available products can be used.

[0025] In the present invention, composition B is prepared by dissolving the above - mentioned anti - fish herpes virus composition in ethanol and then drying. As an implementation method, the composition and ethanol are mixed at 1g:5 - 15ml; as an implementation method, the drying temperature is 40 - 70°C, and the drying time is 1 - 3 hours.

[0026] In the present invention, the composition D is composed of 20 - 40% astragalus extract and 60 - 80% scutellaria extract; as an embodiment, the composition D is composed of 25 - 35% astragalus extract and 65 - 75% scutellaria extract. The preparation method of the astragalus extract or scutellaria extract is the same as that of the above-mentioned danshen extract, dandelion extract, angelica dahurica extract or toosendan fruit extract, which will not be elaborated here. Mix the astragalus extract and scutellaria extract in proportion to obtain the composition D.

[0027] The above-mentioned composition for anti-fish herpesvirus or the compound Chinese herbal medicine preparation for anti-fish herpesvirus of the present invention can be used in the preparation of drugs for preventing and / or treating fish herpesvirus.

[0028] The compound Chinese herbal medicine preparation for anti-fish herpesvirus is scientifically formulated from composition C (containing composition A and B) and composition D, and each component synergistically exerts a multi-level antiviral effect. Composition A takes astragalus, scutellaria and licorice as the core components, in which astragalus polysaccharide and saponins enhance non-specific immunity, baicalein and baicalin directly inhibit virus adsorption and replication, and glycyrrhizic acid interferes with the virus life cycle, jointly constituting a basic antiviral system; composition B obtained by ethanol extraction significantly increases the concentration and bioavailability of active components such as flavonoids and saponins, and strengthens the antiviral effect. Composition C realizes the full-coverage prevention and treatment of different infection stages of herpesvirus through the synergistic effect of the basic immune regulation of composition A and the highly effective antiviral extract of composition B. Composition D further strengthens the antiviral efficacy, its high-concentration astragalus extract enhances the immune response, and the scutellaria extract strongly inhibits virus replication and reduces the inflammatory response. Through the action mechanism of multiple targets and multiple pathways, this compound preparation not only directly inhibits virus replication, but also enhances the immunity of fish, and at the same time has the advantages of not being prone to drug resistance and environmental friendliness, forming a comprehensive prevention and control system that emphasizes both prevention and treatment, and showing good application prospects in the prevention and treatment of fish herpesvirus.

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be described in detail below with reference to the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0030] In the following embodiments, unless otherwise specified, all are conventional methods.

[0031] The Chinese herbal medicines, feeds, antibiotics, grass carps, crucian carps, common carps, mandarin fish, bass, and groupers used in the following embodiments are all common varieties on the market, and can be obtained from commercial channels unless otherwise specified.

[0032] In the following embodiments, unless otherwise specified, the % mentioned in the present invention is by weight percentage.

[0033] In the following embodiments, the preparation methods of the astragalus powder, scutellaria powder, licorice powder and corn starch powder of the present invention are as follows: select high-quality astragalus, scutellaria, licorice or corn as raw materials, rinse them with clean water 3-5 times, place them in an oven at 100 °C for 1-2 hours, break them with a powder mill and sieve them through a sieve with a mesh size of less than 90 meshes.

[0034] Example 1: Composition against fish herpesvirus

[0035] Salvia miltiorrhiza extract 7%, dandelion extract 23%, angelica dahurica extract 30%, toosendan fruit extract 40%.

[0036] The preparation methods of the Salvia miltiorrhiza extract, dandelion extract, angelica dahurica extract and toosendan fruit extract are as follows: select high-quality Salvia miltiorrhiza, dandelion, angelica dahurica or toosendan fruit, rinse them with clean water 5 times, place them in an oven at 80 °C for 2 hours, break them with a powder mill and sieve them through a 200-mesh sieve; mix the powder with ethanol at a weight-to-volume ratio of 1:9, stir for 30 minutes; filter the extract 3 times; concentrate the active ingredients under vacuum conditions with a pressure of 85 Kp and a temperature of 40 °C; place the concentrated solution in an oven at 60 °C for 2 hours to obtain the extract.

[0037] Example 2: Composition against fish herpesvirus

[0038] Salvia miltiorrhiza extract 5%, dandelion extract 10%, angelica dahurica extract 20%, toosendan fruit extract 65%.

[0039] The preparation methods of the Salvia miltiorrhiza extract, dandelion extract, angelica dahurica extract and toosendan fruit extract are as follows: select high-quality Salvia miltiorrhiza, dandelion, angelica dahurica or toosendan fruit, rinse them with clean water 3 times, place them in an oven at 80 °C for 2 hours, break them with a powder mill and sieve them through a 200-mesh sieve; mix the powder with ethanol at a weight-to-volume ratio of 4:6, stir for 30 minutes; filter the extract 3 times; concentrate the active ingredients under vacuum conditions with a pressure of 70 Kp and a temperature of 60 °C; place the concentrated solution in an oven at 50 °C for 4 hours to obtain the extract.

[0040] Example 3: Composition against fish herpesvirus

[0041] Salvia miltiorrhiza extract 10%, dandelion extract 30%, angelica dahurica extract 40%, toosendan fruit extract 20%.

[0042] The preparation methods of Salvia miltiorrhiza extract, Taraxacum mongolicum extract, Angelica dahurica extract, and Toosendan fruit extract are as follows: Select high-quality Salvia miltiorrhiza, Taraxacum mongolicum, Angelica dahurica, or Toosendan fruit, rinse it 3 times with clean water, place it in an oven at 80 °C for 1 hour, use a pulverizer to break it into pieces and pass through a 200-mesh sieve; mix the powder with ethanol at a weight-to-volume ratio of 5:5, and stir for 30 minutes; filter the extract 3 times; concentrate the active ingredients under a vacuum condition with a pressure of 80 Kp and a temperature of 60 °C; place the concentrated solution in an oven at 70 °C for 1 hour to obtain the product.

[0043] Example 4 Compound Chinese Herbal Medicine Preparation Against Fish Herpesvirus

[0044] F1, Mix to obtain Composition A according to the ratio of 10% Astragalus membranaceus powder, 15% Scutellaria baicalensis powder, 20% Glycyrrhiza uralensis powder, and 55% corn starch.

[0045] F2, Dissolve 10 g of the composition in Example 1 in 100 ml of 95% ethanol to obtain Solution B.

[0046] F3, Spray-dry Solution B to obtain Composition B, and mix according to the ratio of 50% Composition A and 50% Composition B to obtain Composition C.

[0047] F4, Mix to obtain Composition D according to the ratio of 40% Astragalus membranaceus extract and 60% Scutellaria baicalensis extract. The preparation methods of Astragalus membranaceus extract and Scutellaria baicalensis extract are the same as those in Example 1.

[0048] F5, Mix according to the ratio of 75% Composition C and 25% Composition D to obtain the compound Chinese herbal medicine preparation against fish herpesvirus.

[0049] Example 5 Compound Chinese Herbal Medicine Preparation Against Fish Herpesvirus

[0050] F1, Mix to obtain Composition A according to the ratio of 15% Astragalus membranaceus powder, 20% Scutellaria baicalensis powder, 30% Glycyrrhiza uralensis powder, and 45% corn starch.

[0051] F2, Dissolve 5 g of the composition in Example 2 in 100 ml of 95% ethanol to obtain Solution B.

[0052] F3, Fluid-bed dry Solution B to obtain Composition B, and mix according to the ratio of 30% Composition A and 70% Composition B to obtain Composition C.

[0053] F4, Mix to obtain Composition D according to the ratio of 20% Astragalus membranaceus extract and 80% Scutellaria baicalensis extract. The preparation methods of Astragalus membranaceus extract and Scutellaria baicalensis extract are the same as those in Example 2.

[0054] F5, Mix according to the ratio of 30% Composition C and 70% Composition D to obtain the compound Chinese herbal medicine preparation against fish herpesvirus.

[0055] Example 6 Compound Chinese Herbal Medicine Preparation Against Fish Herpesvirus

[0056] F1, Mix the astragalus powder at 10%, scutellaria powder at 15%, licorice powder at 20%, and corn starch at 55% to obtain Composition A.

[0057] F2, Dissolve 15 g of the composition in Example 3 in 100 ml of 95% ethanol to obtain Solution B.

[0058] F3, Freeze-dry Solution B to obtain Composition B, and mix Composition A at 60% and Composition B at 40% to obtain Composition C.

[0059] F4, Mix the astragalus extract at 60% and the scutellaria extract at 40% to prepare Composition D. The preparation methods of the astragalus extract and the scutellaria extract are the same as those in Example 3.

[0060] F5, Mix Composition C at 90% and Composition D at 10% to obtain the compound Chinese herbal medicine preparation against fish herpesvirus.

[0061] Example 7

[0062] This example is basically the same as the steps in Example 4, except that in the preparation methods of the danshen extract, dandelion extract, angelica dahurica extract, toosendan fruit extract, astragalus extract, or scutellaria extract, the concentrated solution is placed in an oven at 60°C for 1 hour. By detecting, the content of dihydrochalcone in the active ingredients of the compound Chinese herbal medicine preparation prepared in this step is 15 μg / ml.

[0063] Example 8

[0064] This example is basically the same as the steps in Example 4, except that in the preparation methods of the danshen extract, dandelion extract, angelica dahurica extract, toosendan fruit extract, astragalus extract, and scutellaria extract, the concentrated solution is placed in an oven at 60°C for 3 hours. By detecting, the content of dihydrochalcone in the active ingredients of the compound Chinese herbal medicine preparation prepared in this step is 40 μg / ml.

[0065] Example 9

[0066] This example is basically the same as the steps in Example 4, except that in the preparation methods of the danshen extract, dandelion extract, angelica dahurica extract, toosendan fruit extract, astragalus extract, and scutellaria extract, the concentrated solution is placed in an oven at 60°C for 4 hours. By detecting, the content of dihydrochalcone in the active ingredients of the compound Chinese herbal medicine preparation prepared in this step is 50 μg / ml.

[0067] Example 10

[0068] This example is basically the same as Example 4, except that in the preparation methods of the Salvia miltiorrhiza extract, dandelion extract, Angelica dahurica extract, Toosendan fruit extract, Astragalus membranaceus extract, and Scutellaria baicalensis extract, Salvia miltiorrhiza, dandelion, Angelica dahurica, Toosendan fruit, Astragalus membranaceus, or Scutellaria baicalensis is mixed with ethanol in a ratio of 4:6 and stirred for 30 minutes. The content of chalcone in the active ingredients of the compound Chinese herbal medicine preparation obtained in this step is detected to be 25 μg / ml.

[0069] Example 11

[0070] This example is basically the same as Example 4, except that in the preparation methods of the Salvia miltiorrhiza extract, dandelion extract, Angelica dahurica extract, Toosendan fruit extract, Astragalus membranaceus extract, and Scutellaria baicalensis extract, Salvia miltiorrhiza, dandelion, Angelica dahurica, Toosendan fruit, Astragalus membranaceus, or Scutellaria baicalensis is mixed with ethanol in a ratio of 5:5 and stirred for 30 minutes. The content of umbelliferone in the active ingredients of the compound Chinese herbal medicine preparation obtained in this step is detected to be 25 μg / ml.

[0071] Example 12

[0072] This example is basically the same as Example 4, except that F3 fluidizes and dries Solution B to obtain Composition B, and Composition C is obtained by mixing Composition A and Composition B in a ratio of 50% and 50% respectively.

[0073] Example 13

[0074] This example is basically the same as Example 4, except that F3 freeze-dries Solution B to obtain Composition B, and Composition C is obtained by mixing Composition A and Composition B in a ratio of 50% and 50% respectively.

[0075] Example 14

[0076] This example is basically the same as Example 4, except that F3 rotary flash dries Solution B to obtain Composition B, and Composition C is obtained by mixing Composition A and Composition B in a ratio of 50% and 50% respectively.

[0077] Example 15

[0078] The feeding method of the compound Chinese herbal medicine preparation obtained in Example 4 is to sprinkle the compound Chinese herbal medicine preparation one hour before feeding, and the sprinkling amount is 20 kg / mu.

[0079] Example 16

[0080] The feeding method of the compound Chinese herbal medicine preparation obtained in Example 4 is to feed 20 kg / mu of both the compound Chinese herbal medicine preparation and the feed.

[0081] Example 17

[0082] The feeding method of the compound Chinese herbal medicine preparation prepared in Example 4 is to put a sufficient amount of the medicine in a pocket and hang it in the feed delivery area or the area where the fish must pass for feeding.

[0083] Comparative Example 1

[0084] This comparative example is basically the same as Example 4, except that F1 is missing, and the obtained compound Chinese herbal medicine preparation lacks composition A.

[0085] Comparative Example 2

[0086] This comparative example is basically the same as Example 4, except that F2 is missing and the obtained compound Chinese herbal medicine preparation lacks composition B.

[0087] Comparative Example 3

[0088] This comparative example is basically the same as Example 4, except that F4 is missing and the obtained compound Chinese herbal medicine preparation lacks composition D.

[0089] Comparative Example 4

[0090] The steps of this comparative example are basically the same as those of Example 4, except that in the preparation method of the salvia miltiorrhiza extract, dandelion extract, angelica dahurica extract, Toosendan fruit extract, Astragalus membranaceus extract, and Scutellaria baicalensis extract, salvia miltiorrhiza, dandelion, angelica dahurica, Toosendan fruit, Astragalus membranaceus or Scutellaria baicalensis and ethanol are mixed in a ratio of 9:1 and stirred for 30 minutes. The content of steroidal alkaloids in the active ingredients of the compound Chinese herbal medicine preparation prepared by this step is 25 μg / ml.

[0091] Test Example 1 Inhibitory Effects of Compositions of Examples 4-6 in Different Ratios on Fish Herpes Virus

[0092] (1) Experimental fish: 400 mandarin fish, sea bass, and grouper of size (100±5) g were selected and placed in a pool equipped with oxygenation equipment and severely infected with herpes virus. The pool was divided into four areas, and each area was isolated from other areas. The breeding period was three months.

[0093] (2) Experimental plan: Mandarin fish, seabass and grouper were randomly divided into four groups, with 100 of each fish in each group, and different markings were performed. In total, each group contained 100 mandarin fish, 100 seabass and 100 grouper. Group 1: fed with the compound Chinese herbal preparation of Example 4. Group 2: fed with the compound Chinese herbal preparation of Example 5. Group 3: fed with the compound Chinese herbal preparation of Example 6. Group 4: not fed with the compound Chinese herbal preparation; all groups were fed once at 8 am and 8 pm every day, and the feeding amount was until the fish were full and slightly left over, for three consecutive months. The survival rate of each fish in the four groups after three months is shown in Table 1 below.

[0094] Table 1 Effect of different ratios on fish survival rate

[0095] Group Survival rate of mandarin fish Survival rate of perch Survival rate of grouper The first group 90％ 81％ 87％ The second group 70％ 65％ 72％ The third group 81％ 71％ 61％ The fourth group 50％ 48％ 51％

[0096] The experimental results are shown in Table 1. After three months of feeding, the survival rate of the first group in each experimental group is the highest. The survival rate of mandarin fish reaches 90%, the survival rate of perch reaches 81%, and the survival rate of grouper reaches 87%. The third group is the second, and the second group has a relatively poor effect, but all are higher than the survival rate of the blank control group in the fourth group. This shows that the survival rate of mandarin fish, perch, and grouper after being infected with herpesvirus is lower than 50%. The compound Chinese herbal medicine preparations with different proportions described in the present invention have significantly improved the survival rate of mandarin fish, perch, and grouper, and have an obvious effect of inhibiting fish herpesvirus.

[0097] Test Example 2 Inhibitory effect of Example 4 and Comparative Examples 1-3 with different combination methods of the composition on fish herpesvirus

[0098] (1) Experimental fish: Select 400 mandarin fish, perch, and grouper each with a specification of (100±5) grams, and put them into a pool with oxygenation equipment and serious herpesvirus diseases for feeding. The pool is divided into four areas, and each area is isolated from other areas without being affected. The feeding time is three months.

[0099] (2) Experimental plan: Randomly divide mandarin fish, perch, and grouper into four groups, with 100 fish of each species in each group, and make different marks. Each group contains 100 salmon, 100 perch, and 100 grouper in total. The first group: Feed the compound Chinese herbal medicine preparation made in the steps of Example 4. The second group: Feed the compound Chinese herbal medicine preparation made in the steps of Comparative Example 1. The third group: Feed the compound Chinese herbal medicine preparation made in the steps of Comparative Example 2. The fourth group: Feed the compound Chinese herbal medicine preparation made in the steps of Comparative Example 3; For all groups, feed once at 8:00 am and 8:00 pm every day, and the feeding amount is until they are full with a little remaining. Feed continuously for three months. The survival rates of each fish in the four groups after three months are shown in Table 2 below.

[0100] Table 2 Influence of different combination methods on the survival rates of mandarin fish, perch, and grouper

[0101] Group Survival rate of mandarin fish Survival rate of perch Survival rate of grouper The first group 89％ 80％ 90％ The second group 65％ 55％ 57％ The third group 50％ 61％ 52％ The fourth group 70％ 72％ 65％

[0102] The experimental results are shown in Table 2. After three months of feeding, the survival rate of the first group in each experimental group is the highest. The survival rate of mandarin fish reaches 89%, the survival rate of perch reaches 80%, and the survival rate of grouper reaches 90%. The fourth group is the second, the second group is lower, and the third group has the worst effect. This shows that the compound Chinese herbal medicine preparation prepared by compounding composition C and composition D by mixing composition A and composition B has the best inhibitory effect on the herpesvirus of the three kinds of fish, and the lack of any one composition will reduce the inhibitory effect on the herpesvirus.

[0103] Test Example 3 Inhibitory effect of different dihydrochalcone contents in the Chinese herbal medicine preparation on fish herpesvirus

[0104] The present invention extracts four active ingredients of fish herpes virus resistance, namely dihydrochalcone, chalcone, umbelliferone, and steroidal alkaloids, from the prepared compound Chinese herbal medicine preparation through detection. Each scheme exerts its effects through a multi-target mechanism, such as chalcone and dihydrochalcone synergistic immunomodulation, umbelliferone focusing on relieving inflammation symptoms, and steroidal alkaloids and umbelliferone complementarily inhibiting viruses. Among them, the contents of the four active ingredients contained in the compound Chinese herbal medicine preparations prepared in Example 4, Example 7, Example 8, and Example 9 are shown in Table 3 below.

[0105] Table 3 Composition and efficacy of active ingredients in different examples

[0106]

[0107] It can be seen that the effective ingredients in Example 4 are in the proportion of 25% dihydrochalcone, 30% chalcone, 40% umbelliferone, and 5% steroidal alkaloids, which highlight the direct antiviral effect and are suitable for the acute infection period; the effective ingredients in Example 7 are in the proportion of 15% dihydrochalcone, 30% chalcone, 35% umbelliferone, and 20% steroidal alkaloids, which focus on inhibiting viral replication and quickly relieve infection symptoms; the effective ingredients in Example 8 are in the proportion of 40% dihydrochalcone, 30% chalcone, 10% umbelliferone, and 20% steroidal alkaloids, which can significantly enhance the body's immune defense ability and reduce the risk of infection, and are suitable for long-term health maintenance; the effective ingredients in Example 9 are in the proportion of 50% dihydrochalcone, 10% chalcone, 15% umbelliferone, and 25% steroidal alkaloids, which have both viral inhibition and immune synergistic effects and are suitable for comprehensive protection in daily breeding.

[0108] The test process is as follows:

[0109] (1) Experimental fish: 400 mandarin fish, sea bass, and grouper of size (100±5) g were selected and placed in a pool equipped with oxygenation equipment and severely infected with herpes virus. The pool was divided into four areas, and each area was isolated from other areas. The breeding period was three months.

[0110] (2) Experimental plan: Mandarin fish, seabass and grouper were randomly divided into four groups, with 100 of each fish in each group, and different markings were performed. In total, each group contained 100 salmon, 100 seabass and 100 groupers. Group 1: fed with the compound Chinese herbal preparation prepared in the step of Example 4. Group 2: fed with the Chinese herbal preparation prepared in the step of Example 7. Group 3: fed with the Chinese herbal preparation prepared in the step of Example 8. Group 4: fed with the Chinese herbal preparation prepared in the step of Example 9; all groups were fed once at 8 am and 8 pm every day, and the feeding amount was until the fish were full with a little left over, for three consecutive months. The survival rate of each fish in the four groups after three months is shown in Table 4 below.

[0111] Table 4 Effects of Different Preparation Methods on the Survival Rates of Mandarin Fish, Sea Bass, and Grouper

[0112] Group Survival rate of mandarin fish Survival rate of perch Survival rate of grouper The first group 92％ 83％ 91％ The second group 83％ 77％ 80％ The third group 85％ 83％ 67％ The fourth group 81％ 82％ 84％

[0113] The experimental results are shown in Table 4. After three months of feeding, the survival rate of the first group in each experimental group was the highest, with the survival rate of mandarin fish reaching 92%, the survival rate of sea bass reaching 83%, and the survival rate of grouper reaching 91%. The third group was the second, the fourth group was lower, and the second group had the worst effect. This indicates that the active ingredients of the compound Chinese herbal medicine preparation prepared by the Chinese herbal medicine preparation method in Example 4 of this application include dihydrochalcone, chalcone, umbelliferone, and steroidal alkaloids, and when the content of the active ingredient dihydrochalcone in the compound Chinese herbal medicine preparation is 15 - 50 μg / ml, it all shows an inhibitory effect on fish herpesvirus.

[0114] Test Example 4 Inhibitory Effects of Different Extraction Methods in Example 4, Example 10 - 11, and Comparative Example 4 on Fish Herpesvirus

[0115] By detecting the compound Chinese herbal medicine preparation, it is found that the four active ingredients contained in the compound Chinese herbal medicine preparations prepared in Example 4, Example 10, Example 11, and Comparative Example 4 are as shown in Table 5 below.

[0116] Table 5 Composition and Efficacy of Active Ingredients in Different Examples and Comparative Examples

[0117]

[0118] Thus, it can be seen that in Example 4, the active ingredients are in the proportion of 25% dihydrochalcone, 30% chalcone, 40% umbelliferone, and 5% steroidal alkaloids, highlighting the direct antiviral effect and being suitable for the acute infection period; in Example 10, the active ingredients are in the proportion of 15% dihydrochalcone, 25% chalcone, 40% umbelliferone, and 20% steroidal alkaloids, mainly strengthening immune regulation and being suitable for preventive health care; in Example 11, the active ingredients are in the proportion of 30% dihydrochalcone, 10% chalcone, 25% umbelliferone, and 35% steroidal alkaloids, balancing the antiviral and immune regulation functions and being suitable for conventional farming; in Comparative Example 4, the active ingredients are in the proportion of 50% dihydrochalcone, 10% chalcone, 15% umbelliferone, and 25% steroidal alkaloids, being anti - inflammation - dominant.

[0119] The test process is as follows:

[0120] (1) Experimental fish: Select 400 mandarin fish, sea bass, and grouper each with a specification of (100 ± 5) grams, and put them into a pool with aeration equipment and severe herpesvirus disease for feeding. The pool is divided into four areas, and each area is isolated from the other areas without being affected. The feeding time is three months.

[0121] (2) Experimental scheme: Randomly divide mandarin fish, perch, and grouper into four groups, with 100 fish of each species in each group. Different markings are made, so each group contains 100 salmon, 100 perch, and 100 grouper. Group 1: Feed the compound Chinese herbal medicine preparation made in step of Example 4. Group 2: Feed the Chinese herbal medicine preparation made in step of Example 10. Group 3: Feed the Chinese herbal medicine preparation made in step of Example 11. Group 4: Feed the Chinese herbal medicine preparation made in step of Comparative Example 4; For all groups, feed once at 8:00 am and once at 8:00 pm every day, and the feeding amount is until they are full with a little remaining. Do this continuously for three months. The survival rates of each type of fish in the four groups after three months are shown in Table 6 below.

[0122] Table 6 Effects of different extraction methods on the survival rates of mandarin fish, perch, and grouper

[0123]

[0124]

[0125] The experimental results are shown in Table 6. After three months of breeding, the survival rate of Group 1 in each experimental group is the highest, with the survival rate of mandarin fish reaching 92%, the survival rate of perch reaching 83%, and the survival rate of grouper reaching 91%. The second group is the second, the third group is lower, and the fourth group has the worst effect. This shows that the extraction ratio of Chinese herbal medicine to ethanol in the preparation method of Chinese herbal medicine in Example 4 of this application will change the content of active ingredients in Chinese herbal medicine. The compound Chinese herbal medicine preparation prepared with an extraction ratio of Chinese herbal medicine to ethanol of 1:9 - 5:5 has an inhibitory effect on fish herpes virus, while the compound Chinese herbal medicine preparation prepared with an extraction ratio of Chinese herbal medicine to ethanol of 9:1 has no inhibitory effect on fish herpes virus.

[0126] Test Example 5 Inhibitory effects of different drying methods in Example 4, Examples 12 - 14 on fish herpes virus

[0127] (1) Experimental fish: Select 400 mandarin fish, perch, and grouper each with a specification of (100 ± 5) grams, and put them into a pool with oxygenation equipment and serious herpes virus diseases for breeding. The pool is divided into four areas, and each area is isolated and not affected by other areas after isolation. The breeding time is three months.

[0128] (2) Experimental scheme: Randomly divide mandarin fish, perch, and grouper into four groups, with 100 fish of each species in each group. Different markings are made, and each group contains 100 salmon, 100 perch, and 100 grouper in total. The first group: Feed the compound Chinese herbal medicine preparation made in step of Example 4. The second group: Feed the Chinese herbal medicine preparation made in step of Example 12. The third group: Feed the Chinese herbal medicine preparation made in step of Example 13. The fourth group: Feed the Chinese herbal medicine preparation made in step of Example 14; For all groups, feed once at 8:00 am and 8:00 pm every day, and the feeding amount is until they are full with a little remaining. Do this continuously for three months. The survival rates of each type of fish in the four groups after three months are shown in Table 7 below.

[0129] Table 7 Effects of different drying methods on the survival rate of fish

[0130] Group Survival rate of mandarin fish Survival rate of perch Survival rate of grouper The first group 90％ 85％ 91％ The second group 78％ 78％ 85％ The third group 81％ 82％ 79％ The fourth group 65％ 68％ 81％

[0131] The experimental results are shown in Table 7. After three months of feeding, the survival rate of the first group in each experimental group is the highest, with the survival rate of mandarin fish being 90%, the survival rate of perch being 85%, and the survival rate of grouper being 91%. The third group is the second, the second group is lower, and the fourth group has the worst effect. This shows that the drying method of solution B in this application will affect the inhibitory effect of the compound Chinese herbal medicine preparation on herpes virus, but the compound Chinese herbal medicine preparations prepared by different drying methods all have an inhibitory effect on fish herpes virus.

[0132] Test Example 6 Inhibitory effects of Examples 15 - 17 with different feeding methods on fish herpes virus

[0133] Feed normal feed as the control group.

[0134] (1) Experimental fish: Select 400 mandarin fish, perch, and grouper each with a specification of (100 ± 5) grams, put them into a pool with oxygenation equipment and serious herpes virus diseases for breeding. The pool is divided into four areas, and each area is isolated and not affected by other areas after isolation. The breeding time is three months.

[0135] (2) Experimental scheme: Randomly divide mandarin fish, perch, and grouper into four groups, with 100 fish of each species in each group. Different markings are made, and each group contains 100 salmon, 100 perch, and 100 grouper in total. The first group: Feed according to the method of Example 15. The second group: Feed according to the method of Example 16. The third group: Feed according to the method of Example 17. The fourth group: Feed according to the method of the control group; For all groups, feed once at 8:00 am and 8:00 pm every day, and the feeding amount is until they are full with a little remaining. Do this continuously for three months. The survival rates of each type of fish in the four groups after three months are shown in Table 8 below.

[0136] Table 8 Effects of different feeding methods on the survival rates of mandarin fish, perch, and grouper

[0137] Group Survival rate of mandarin fish Survival rate of perch Survival rate of grouper The first group 88％ 90％ 95％ The second group 80％ 78％ 81％ The third group 75％ 61％ 81％ The fourth group 51％ 49％ 56％

[0138] The experimental results are shown in Table 8. After three months of feeding, the survival rate of the first group in each experimental group is the highest, with the survival rate of mandarin fish being 88%, the survival rate of sea bass being 90%, and the survival rate of grouper being 95%. The second group is the second, the third group is lower, and the fourth group has the worst effect. This shows that the compound Chinese herbal medicine preparation prepared in Example 4 of the present invention has different inhibitory effects on herpes virus when fed in different feeding methods, but has inhibitory effects on the herpes virus of all three kinds of fish, and is higher than the survival rate without feeding the compound Chinese herbal medicine preparation.

[0139] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection of the present invention.

Claims

1. A composition for resisting fish herpes virus, characterized in that: The invention comprises the following components in weight percentage: 5-10% of salvia miltiorrhiza extract, 10-30% of dandelion extract, 20-40% of angelica dahurica extract and 20-65% of toosendan fruit extract.

2. The composition according to claim 1, characterized in that The preparation method of the salvia miltiorrhiza extract, dandelion extract, angelica dahurica extract or toosendan fruit extract is as follows: powder of salvia miltiorrhiza, dandelion, angelica dahurica or toosendan fruit and ethanol are mixed in a mass volume ratio of 1:9-5:5 g / cm 3 Extraction, concentration and drying are performed.

3. The composition according to claim 2, characterized in that The drying method includes spray drying, fluidized bed drying, freeze drying or spin flash drying.

4. The composition according to claim 2 or 3, characterized in that The drying temperature is 50-70° C., and the drying time is 1-4 hours.

5. A compound Chinese herbal medicine preparation for resisting fish herpes virus, characterized in that: The composition comprises the following weight percentages: 30-90% of composition C and 10-70% of composition D; Taking the mass of composition C as 100%, the composition C consists of 30-60% of composition A and 40-70% of composition B; Taking the mass of composition A as 100%, the composition A is composed of 5-15% astragalus powder, 10-20% scutellaria powder, 10-30% liquorice powder and 45-75% corn starch; the composition B is prepared by dissolving the composition according to any one of claims 1 to 3 in ethanol and then drying; Taking the mass of composition D as 100%, the composition D consists of 20-60% of astragalus extract and 40-80% of scutellaria baicalensis extract.

6. The compound Chinese herbal medicine preparation according to claim 5, characterized in that: The compound Chinese herbal medicine preparation consists of 60-90% of composition C and 10-40% of composition D.

7. The compound Chinese herbal medicine preparation according to claim 5, characterized in that: The composition D consists of 20-40% of Astragalus membranaceus extract and 60-80% of Scutellaria baicalensis extract.

8. The compound Chinese herbal medicine preparation according to claim 5 or 7, characterized in that: The preparation method of the astragalus extract and the scutellaria extract is as follows: the powder of astragalus or scutellaria is mixed with ethanol in a mass volume ratio of 1:9-5:5 g / cm 3 Extraction, concentration and drying are performed.

9. A method for preparing the anti-fish herpes virus compound Chinese herbal medicine preparation according to any one of claims 5 to 8, characterized in that: Composition A is mixed with composition B to obtain composition C, and composition C is mixed with composition D to obtain composition C.

10. Use of the composition according to any one of claims 1 to 4 or the compound Chinese herbal medicine preparation according to any one of claims 5 to 8 in the preparation of a drug for preventing and / or treating fish herpes virus disease.

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