JP2001122796A - Antiviral substance, method for extracting the same, its formula feed and method for prophylaxis and treatment of viral infectious disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antiviral substance having high infection prophylactic effects on viral infectious diseases and simply producible from a naturally occurring raw material at a low cost, to provide a method for extracting the antiviral substance, to obtain a formula feed thereof and to provide a method for the prophylaxis of the viral infectious diseases. SOLUTION: This antiviral substance consists essentially of a substance extracted from Eisenia bicyclis Setchell which is one species of seaweeds with an alcohol. Thereby, the antiviral substance having high infection prophylactic effects on the viral infectious diseases can simply be produced from the naturally occurring raw material at a low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antiviral substance capable of preventing viral infections in aquaculture animals and livestock animals, and more particularly to an antiviral substance extracted from seaweed alame.

[0002] In recent years, in the aquaculture industry and the livestock industry, various virus infections have occurred in aquaculture animals and livestock animals, resulting in industrial damage and pressure on the management of these industries. The main virus infectious diseases are aquaculture fish, mainly red sea bream, red sea bream, blue sea bream, yellow sea bream, yellowtail, flyfish, amberjack, eastern horse mackerel, etc., and viral neuronecrosis of Japanese flounder and horse mackerel. (VNN), acute shrimp viremia (PA
V), stomatosis of tiger puffer, infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) of salmonid fish. Among these viral infections, red sea bream iridovirus infection and acute viremia occur annually at farms in western Japan and cause tremendous damage. Examples of livestock animals include Aujeszky's disease in pigs, Akabane's disease in cattle, Ainovirus disease, Cheuzin's disease, Newcastle disease in chickens, and Reovirus disease. This time, by adding an antiviral substance extracted from alame to a mixed feed and administering it to target animals for a certain period of time, it is possible to prevent viral infections in aquaculture animals and livestock animals, It can contribute to stable management.

[0003]

2. Description of the Related Art Conventionally, as a preventive measure against this kind of virus infection, a method of enhancing immunity against virus infection by administering vitamins, immunostimulants, vaccines and the like to aquaculture animals and livestock animals. I got it. The vitamin preparation and the immunostimulant are mixed and administered to the feed of aquaculture animals and livestock animals, and the vaccine is directly inoculated to aquaculture animals and livestock animals.

[0004]

The conventional preventive measures against viral infections have been carried out as described above. Therefore, vitamins and immunostimulants, etc., enhance the inherent immunity of aquaculture animals and livestock animals. This is intended to be improved by the contained feed and does not directly act on the virus, and thus has a problem that its effect of preventing infection is not sufficient.

[0005] In addition, the vaccine must be acquired by directly inoculating aquaculture animals, so that the technique of vaccination must be acquired, and the target aquaculture animals must be inoculated one by one. A large labor burden is required. In addition, vaccines are expensive and used in large quantities, which increases the cost burden and deteriorates the business balance. further,
In recent years, with the increasing number of consumers who dislike the use of vaccines and the like, measures against viral infectious diseases that do not use vaccines are also required.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and has an antiviral substance which has a high effect of preventing viral infection and can be easily and inexpensively produced from natural raw materials. An object of the present invention is to provide the compound feed and a method for preventing a virus infection.

[0007]

The antiviral substance according to the present invention is mainly composed of a substance obtained by extracting alcohol from alame. As described above, in the present invention, since a substance obtained by alcohol extraction from alame which is a kind of seaweed is used as a main component, it is possible to easily and inexpensively produce an antiviral substance having a high effect of preventing viral infection from natural raw materials. Can be.

[0008] The method for extracting an antiviral substance according to the present invention is obtained by adding a sterilized phosphate buffered saline to alame to form a mixture, forming a homogenate of the mixture, and then centrifuging the homogenate. Alcohol is added to the precipitate to form an alcohol mixture, a homogenate of the alcohol mixture is produced, and the supernatant obtained by centrifuging the homogenate is concentrated. As described above, in the present invention, since a plant material produced from alame is used, it can be grown under the same growth conditions as a natural animal against a virus which does not cause the disease in the natural animal.

In the method for extracting an antiviral substance according to the present invention, sterilized phosphate buffered physiological saline is added in an amount of 1 to 10 parts by weight per 1 part by weight of alame, if necessary. As described above, in the present invention, the extraction of the antiviral substance can be facilitated, and the antiviral substance can be produced at an appropriate concentration. In the method for extracting an antiviral substance according to the present invention, an alcohol is added to 1 part by weight of
It is added in the range of 0 parts by weight. As described above, in the present invention, the extraction of the antiviral substance can be facilitated, and the antiviral substance can be produced at an appropriate concentration.

[0010] The feed containing the antiviral substance according to the present invention comprises:
An extract of an antiviral substance obtained by alcohol extraction from 10 kg (wet weight) of alame is added to a mixed feed of 60 to 150 kg. The antiviral substance-containing feed according to the present invention may be in the form of powder, granules or pellets as required. As described above, in the present invention, by forming a feed form in a predetermined state, when mixing with other live foods, a feed suitable for the administration form can be selected, and mixing can be facilitated.

The method for preventing and treating viral infection according to the present invention comprises administering an amount corresponding to 2 to 4% by weight of the body weight of the animal to be prevented for preventing viral infection. The method for preventing and treating viral infectious diseases according to the present invention is such that the administration period is at least 30 days as necessary.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (First Embodiment of the Present Invention) Hereinafter, an antiviral substance according to a first embodiment of the present invention will be described with reference to FIG. 1 together with a method for extracting the same. FIG. 1 shows an operation flowchart in the method for extracting an antiviral substance according to the present embodiment.

The antiviral substance according to the present embodiment is a substance obtained by alcohol extraction from alame which is a seaweed of brown algae, and has an anti-iridovirus activity. Next, a method for extracting an antiviral substance having the above-mentioned properties from Alameda is described. First, the slime was washed with sterilized phosphate buffered saline (hereinafter, PBS (-)) to remove mucus and deposits on the surface of the slime (step 1). 4 times (40
Kg) PBS (-) is added (step 2). This PB
S (-) can be added in the range of 1 to 10 parts by weight based on 1 part by weight of alame.

A homogenate is produced by homogenizing the mixture obtained by adding PBS (-) to the larks at 7,500 [rpm] for 5 minutes using a homogenizer (step 3), and this homogenate is 3,000 [rpm].
For 5 minutes (Step 4) to complete the PBS extraction step. Further, an alcohol extraction step is performed on the precipitate obtained in the PBS extraction steps 1 to 4. In this alcohol extraction step, four times the amount of ethanol (99.5%) is added to the precipitate to produce an ethanol mixture (step 5). This ethanol mixture was subjected to 7,500 [r] using a homogenizer.
pm] to produce a homogenate of ethanol (step 6).

The homogenate of ethanol was added to 3,000
The mixture is centrifuged at 0 [rpm] for 5 minutes (step 7), and the supernatant obtained by the centrifugation is concentrated under reduced pressure to 3 l (step 8). The concentrated supernatant serves as an antiviral substance of the alame extract. The antiviral substance, which is an ethanol extract of alame thus obtained, has the following chemical properties. First, it shows solubility in common organic solvents and can be easily extracted from alcohols such as ethanol, especially after extraction with a buffer solution. Second 10
Heating at 0 ° C. for 10 minutes does not inactivate the antiviral activity. Third, after ultrafiltration through an ultrafiltration membrane whose molecular weight cuts off 3,000, antiviral activity is present in the filtrate.

The antiviral substance of the present invention comprising an ethanol extract of alame having antiviral activity may be itself or a further concentrated form thereof, or a solid form obtained by drying.

As described above, a plant-based antiviral substance containing chlorophyll can be produced by simply extracting from alame which is a kind of brown algae vastly existing in the ocean. When the target animal is red sea bream, it is possible to improve the antiviral prophylactic and therapeutic properties of cultured red sea bream, even against red sea bream iridovirus, which is rarely infected by natural red sea bream,
The cost of tail administration can be around 7 yen. On the other hand, in the case of a red sea bream iridovirus vaccine, a cost of 35 yen is required for one red sea bream red fish, and the present embodiment can achieve a significant cost reduction. In addition, since the antiviral substance according to the present embodiment is produced from a botanical substance contained in natural alame, it can be claimed that there is no phytotoxicity, side effects, etc. even for consumers who seek the target animal for food. .

In the above embodiment, ethanol is used in the alcohol extraction step in the extraction method.
The extraction operation can be performed using alcohol such as methanol. In addition, although the homogenizer is used in the generation of the homogenate in the extraction method of the above embodiment, other crushing means such as a Waring blender may be used.

(Second Embodiment of the Present Invention) The second embodiment of the present invention
10 kg of the antiviral substance-containing feed according to the embodiment
And an extract of the antiviral substance produced according to the first embodiment from Alame is added to 60 to 150 kg of the compound feed. The formulated feed to which this antiviral substance is added can be formed into powder, granules or pellets, and can be formed into various shapes depending on the type of animal to be prevented for preventing viral infection or its use. can do.

The weight of the alame and the compound feed is
The antiviral substance-containing feed can be produced at a ratio where the weight of the compound feed is 6 to 12 times the weight of the alame. Next, a method for preventing a viral infectious disease using an antiviral substance-containing feed based on the above configuration will be described. First, the body weight of the animal to be prevented is measured, and an antiviral substance-containing feed equivalent to the body weight is administered to the animal to be prevented. The administration to the animal to be prevented is performed for at least 30 consecutive days.

As described above, since the antiviral substance-containing feed according to the present embodiment can be added with a plant antiviral substance containing chlorophyll extracted from alame at the stage of producing the compound feed, the antiviral substance-containing feed can be added. Can be produced as a feed to which is added. Therefore, it can be easily and reliably administered once to a large number of animals to be prevented as a part of daily feeding work to cultured aquatic animals, livestock animals, etc. which are the animals to be prevented.

[0022]

Examples Examples 1 to Examples are experimental examples in which a method for preventing and treating viral infections is carried out using an antiviral substance and an antiviral substance-containing feed extracted and manufactured based on the above embodiments. 7 will be described separately.

Example 1 The antiviral activity of the antiviral substance according to this embodiment was tested. This test method is based on the plaque reduction method (Kamei et al., 198
8) Made by. 200 µl of infectious hematopoietic necrosis virus (IHNV) solution of salmonid fish (about 200 PFU) and 200 µl of antiviral substance which is an ethanol extract of sea squid
and the reaction mixture was reacted at 15 ° C. for 1 hour. The reaction mixture was confluent in a 24-well plate and cultured for one day in cultured fish cells (CHSE-214).
Was inoculated into 2 wells at 200 μl each. After this inoculation, the virus was allowed to adsorb to the cells at 15 ° C. for 1 hour, and then a 0.8% methylcellulose medium (Kamei et al., 1
987) and cultured at 15 ° C. for 7 days.

After culturing, the cells were fixed with 10% formalin and then stained with 0.1% crystal violet. Using the stained cell sheet as a background, the number of clear plaques was counted,
Plaque reduction was observed. The plaque reduction rate is 2w each
The average value of ell was determined. In the test results obtained in the above examples, plaque reduction with a virus killing rate of 100% was obtained.

(Example 2) The characteristics of heat stability of the antiviral substance according to the present embodiment were tested. In this test method, an antiviral substance extracted from alame with ethanol was heat-treated at 30 to 100 ° C., and then the antiviral activity was examined by the plaque reduction method in the same manner as in the test of Example 1. The test results obtained by this example are:
It was found that the antiviral activity of the antiviral substance was not reduced or deactivated by the heat treatment with carohydrate up to 100 ° C., and was stable to heat.

Example 3 Antivirus according to the present embodiment
Red sea bream ylide comparing substances to vitamins and immunostimulants
The virus attack properties (preventive effect) were tested. This
Test method differs for each test area in fish farms
Additives (antiviral substance according to this embodiment, vitamin C
And E, immunostimulants) in different combinations
Feeding the red sea bream fish for two months on a mixed feed, each test
Red sea bream fish (FL = 5cm)
(15 ° C) in a water tank (volume: 160 l)
Accepted. Next, red sea bream virus (TCID)₅₀=
10_{3.0 Five}-10_3.8) Was injected intraperitoneally 0.1 ml
Thereafter, the animals were bred and observed without feeding for 17 days to determine the survival rate. Ma
Red sea bream killer fish virus test (monochrome
Null fluorescent antibody method), general bacterial test (BHI medium, 25
C. for 48 hours) and a parasite test.

FIG. 2 shows the test results obtained in this example. The survival rate of the test red sea bream in the test group to which the antiviral substance-containing feed to which the antiviral substance, which is an ethanol extract of larva, was added was administered. As shown in FIG. 2, the survival rate of the test red sea bream in the test group to which the compound feed containing this substance and vitamin C and E was administered was 76% on the 17th day. It was higher than the test plot. As a result of examination of dead fish, red sea bream iridovirus was detected in all individuals, and no bacteria and parasites were observed.

(Example 4) The aggressive properties (preventive effect) against red sea bream iridovirus of the feed containing the antiviral substance having different concentrations of the antiviral substance according to the present embodiment were tested. In this test method, anti-viral substances differing in the concentration of added anti-viral substance (concentration = 1/1, 1/3, 1/6, 1/12) in ethanol extract of alame for each test plot in a fish farm. Red sea bream red fish were reared for 2 months on a mixed feed. This concentration 1/1 corresponds to 3% of the weight of the red sea bream fish. Next, 15 red sea bream fish (FL = 5 cm) from each test group were housed in a water tank (volume: 160 l) in an indoor constant temperature laboratory (25 ° C.), and red sea bream iridovirus (TCID ₅₀ = 10 _4.5 to 10) was stored. _5.55 ) was injected intraperitoneally in an amount of 0.1 ml, and the animals were bred and observed without feeding for 17 days to determine the survival rate. In addition, red sea bream iridovirus test (monoclonal fluorescent antibody method), general bacterial test (BHI medium, cultured at 25 ° C. for 48 hours) and parasite test of dead fish were performed.

The test results obtained in this example show that the survival rate of red sea bream in the test plot to which the antiviral substance-containing feed, which was an ethanol extract of alame, was added at a concentration of 1/1, was administered. As shown in FIG. As a result of examination of dead fish, red sea bream iridovirus was detected in all individuals, and no bacteria and parasites were observed.

Example 5 The aggressive properties (preventive effect) against red sea bream iridovirus due to differences in the administration period of the antiviral substance-containing feed to which the antiviral substance according to the present embodiment was added were tested. In this test method, an antiviral substance-containing feed supplemented with an antiviral substance, which is an ethanol extract of alame, was added to a fish farm at each test area for 15 days.
After feeding for 30, 45 and 60 days, the red sea bream was reared for 2 months. Next, 15 fish fry (FL = 5 cm) from each test plot were each housed in a water tank (volume: 160 l) in an indoor constant temperature laboratory (25 ° C.), and red sea bream virus (TC
ID ₅₀ = 10 _3.25 ) was injected intraperitoneally 0.1 ml and then 1
The animals were bred and observed for 7 days without feeding to determine the survival rate. In addition, red sea bream virus testing of dead fish (monoclonal fluorescent antibody method) and general bacterial testing (BHI medium, 25 ° C, 4 ° C)
8 hours) and a parasite test. Furthermore, using the spleen of the surviving test red sea bream, red sea bream iridovirus test (monoclonal fluorescent antibody method) was performed.

The test results obtained in this example show that the survival rate of the test red sea bream in the test group fed the antiviral substance-containing feed supplemented with the antiviral substance, which is an ethanol extract of larva, for 60 days is shown in FIG. As shown in FIG. 4, the survival rate of the test red sea bream in the test group administered for 30 days and 45 days was the highest at 84% on the 17th day, and was 80% on the 17th day as shown in FIG.
% Was high. As a result of examination of dead fish, red sea bream iridovirus was detected in all individuals, and no bacteria and parasites were observed.

Example 6 Antivirus according to the present embodiment
After administering the compound feed and injecting the antiviral substance
Fate characteristics of red sea bream iridovirus due to differences in elapsed days
Was tested. This test method is used for fish farms.
An antiviral substance, an ethanol extract of rice, was added.
Feed antiviral substance-containing feed for 30 days and feed red sea bream
Bred. These red sea bream fish (FL = 5cm)
20 in a water tank (capacity: 160 l) in a constant temperature laboratory (25 ° C)
Tail housed, red sea bream iridovirus (TCID)₅₀= 10
_3.25) Was intraperitoneally injected 0.1 l after 17 days without feeding
Bred. Next, from the day after the injection, take two fish every two days.
For red sea bream virus testing using spleen
Clonal fluorescent antibody method).

The test results obtained by this embodiment are:
As shown in FIG. 5, after injection of red sea bream virus into the test red sea bream, red sea bream virus was not detected on the first and third days, but was detected on the fifth, seventh and ninth days, but was detected on the eleventh day. No subsequent detection. From this fact, injection of antiviral substance and administration of antiviral substance-containing feed have a therapeutic effect on red sea bream infected by killing red sea bream after infection, even if red sea bream is infected with red sea bream iridovirus. I understand.

(Example 7) The effects of the administration of the antiviral substance-containing feed according to the present embodiment on the prevention of infection and the growth were tested in the field. This test method
The test is carried out by comparing an antiviral substance-containing feed with an antiviral substance, which is an ethanol extract of alame, with a non-added compound feed.

First, at a fish farm, a feed containing an antiviral substance and a feed without additives were fed, and the juvenile red sea bream was raised from June to October. Survival rate after the occurrence of red sea bream iridovirus infection during the period was examined, and red sea bream fish was examined for red sea bream virus (monoclonal fluorescent antibody method), common bacteria (BHI medium, 25 ° C, culture for 48 hours) and parasite tests. Carried out. The body length and weight were measured twice a month.

The test results obtained in this example indicate that red sea bream iridovirus infection occurred from late August to late September. As shown in FIG. 6, the survival rate of the red sea bream fish in each test group at the end of this infection was 98% in the test group fed with the antiviral substance-containing feed to which the antiviral substance, which was an ethanol extract of alame, was added. %, Compared with 83% in the test group fed the additive-free feed. As a result of examination of the dead red sea bream, the red sea bream iridovirus was detected from all the individuals, and no bacteria and parasites were observed. In addition, as shown in FIG. 7, there was no difference in body length and weight of the red sea bream in each test group at the end of the test, indicating that the effect of the antiviral substance-containing feed on red sea bream growth was not recognized.

[0037]

According to the present invention, an antiviral substance having a high effect of preventing infection of a virus infection is easily and inexpensively produced from natural raw materials since the main ingredient is a substance obtained by extracting alcohol from alame which is a kind of seaweed. It has the effect that it can be done. Further, in the present invention, since it is a plant material produced from alame, it has an effect that it can be grown against viruses that do not cause disease in natural animals under the same growth conditions as natural animals.

Further, the present invention has an effect that the extraction of the antiviral substance can be facilitated and the antiviral substance can be produced at an appropriate concentration. Furthermore, in the present invention, by forming the feed form in a predetermined state, when mixing with other live foods, it is possible to select one suitable for the administration form, and there is an effect that mixing can be facilitated.

[Brief description of the drawings]

FIG. 1 is an operation flowchart of an antiviral substance extraction method according to a first embodiment of the present invention.

FIG. 2 is a characteristic chart of the preventive effect on red sea bream iridovirus in a test comparing an antiviral substance with a vitamin / immunostimulant in Example 3 of the present invention.

FIG. 3 is a characteristic diagram of a preventive effect test on red sea bream iridovirus due to a difference in the addition concentration of an antiviral substance in Example 4 of the present invention.

FIG. 4 is a characteristic diagram of a preventive effect test on red sea bream iridovirus due to a difference in administration period of an antiviral substance-containing feed in Example 5 of the present invention.

FIG. 5 is a table showing the results of a virus detection test after inoculation of red sea bream virus in Example 6 of the present invention.

FIG. 6 is a characteristic diagram of the effect of preventing infection after administration of an antiviral substance-containing feed in Example 7 of the present invention.

FIG. 7 is a characteristic diagram of an effect test on growth after administration of an antiviral substance-containing feed in Example 7 of the present invention.

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 2B150 AA01 AA07 AA08 AB10 CE26 DD48 DD57 4C088 AA13 AC15 BA10 CA06 CA12 MA36 MA41 MA43 NA14 ZB33 ZC61 ZC65 4H055 AA01 AA02 AA03 AB10 AB29 AC60 AD15 AD22 AD30 CA61

Claims (8)

[Claims] 1. An antiviral substance comprising, as a main component, a substance extracted from alame with alcohol. 2. A mixture is prepared by adding sterilized phosphate buffered saline to alame to form a homogenate of the mixture, and thereafter, the alcohol mixture is added to a precipitate obtained by centrifuging the homogenate to form an alcohol mixture. A method for extracting an antiviral substance, comprising producing a homogenate of the alcohol mixture, and concentrating a supernatant obtained by centrifuging the homogenate. 3. The method for extracting an antiviral substance according to claim 2, wherein 1 part by weight of alame is added with 1 to 10 parts by weight of sterilized phosphate buffered saline. How to extract viral material. 4. The method for extracting an antiviral substance according to claim 2, wherein the alcohol is added in an amount of 1 to 10 parts by weight per 1 part by weight of the precipitate. How to extract the substance. 5. An antiviral substance extract obtained by alcohol extraction from 10 kg (wet weight) of alame is used for 60 to 150 k.
g. a feed containing an antiviral substance, wherein the feed is added to the feed containing g. 6. The feed containing the antiviral substance according to claim 5, wherein the feed is in the form of powder, granules or pellets. 7. A feed comprising the antiviral substance-containing feed according to claim 5 or 6 in an amount corresponding to 2 to 4% by weight of the body weight of the animal to be prevented for preventing viral infection. How to prevent and treat viral infections. 8. The method for preventing and treating viral infection according to claim 7, wherein the administration period is at least 30 days.