WO2006093164A1

WO2006093164A1 - Virus infection and proliferation inhibitor containing earthworm-derived component

Info

Publication number: WO2006093164A1
Application number: PCT/JP2006/303786
Authority: WO
Inventors: Mitsuhiro Ueda; Satoshi Ohki; Keiji Mine
Original assignee: Osaka Prefecture
Priority date: 2005-02-28
Filing date: 2006-02-28
Publication date: 2006-09-08
Also published as: JPWO2006093164A1

Abstract

It is intended to provide a pharmaceutical, an agrochemical, an animal feed or the like that has an action to inhibit virus infection and proliferation and contains an earthworm-derived active ingredient with, for example, a molecular weight of 10,000 or more and/or an absorbance feature at 280 nm.

Description

Specification

Viral infection and growth inhibitor containing earthworm-derived components

Technical field

[0001] The present invention relates to a drug, agrochemical, or the like, which contains an earthworm-derived component as an active ingredient, and has a virus infection and virus growth inhibitory action.

Background art

Animal cells and body fluids contain many polymer substances as constituents, and the chemical composition of these polymer substances differs depending on the animal species. Each living organism has its own chemical identity. Different types of macromolecules may enter the living body as they are from the outside, viruses with different types of polymer on their surfaces may invade, and their own abnormal constituents may appear. The role of the immune system is to protect the living body from such exogenous and endogenous different macromolecules. The immune system consists of lymphoid organs, lymphoid tissues found in non-lymphatic organs, lymphocytes contained in blood and lymph, lymphocytes and plasma cells scattered throughout the connective and epithelial tissues throughout the body. Lymphocytes continuously circulate through each component of the immune system and exert defense power.

[0003] Higher organisms protect themselves by the immune system described above. It is presumed that lower organisms that do not have lymph have their own self-defense system. We have focused on earthworms among the organisms classified as lower organisms, and have clarified their self-defense systems.

As an example of antibacterial substances, antibacterial peptides derived from insect body fluids are known (Patent Document 1).

However, the antibacterial spectrum of antibacterial peptides derived from insect body fluids is S. aureus

It is limited to MRSA, Escherichia coli, and Pseudomonas aeruginosa, and no antibacterial activity against viruses has been described.

[0004] Compared to human bacterial diseases, viral diseases are relatively insensitive to current chemotherapy. However, human beings can produce effective immunity against natural viral diseases with vaccines. All viral coat proteins can be antigens, except HIV Thus, it is possible to make vaccines against all viruses cultured in the laboratory. But for many viral diseases, this approach has its limitations.

[0005] The common cold is a syndrome caused by viral infection of the upper respiratory tract. Viruses that cause upper respiratory tract infection are influenza classified as the myxovirus group, parainfluenza classified as the paramyxowinore group, and coronavirus. Coxsackie viruses, echoviruses, and rhinoviruses, which are classified into groups, reoviruses, adenovirus groups, and picornavirus groups, are well known. In particular, influenza is a threat to mankind, and the danger of avian influenza has been widely known in recent years.

[0006] Conventionally, earthworms are used as fishing baits, for soil improvement, for garbage disposal, depending on their characteristics, and have been used in various fields. Bioactive proteins have been isolated, and physiological and biochemical studies have been conducted for their use.

As Table 1 shows the names of bioactive proteins derived from earthworms, their molecular weights, and physiological actions, these proteins have been proved to have many useful physiological actions. A method for producing a health supplement using earthworms as a raw material has also been proposed (Patent Document 2).

[0007] [Table 1]

Earthworm-derived bioactive protein

Protein name Molecular weight Physiological action

Hetidine 40, 000 hemolytic activity, antibacterial activity, blood coagulation activity Eiseniapore 38, 000 hemolytic activity

CCF-1 (Cytolytic factor 1) 42, 000 β -1,3 Glucan and lipopolysaccharide binds Lysenin 41,000 Specific binding to sphingomyelin In Chinese medicine, known as terrestrial dragon, has been drunk for a long time, It is known to be safe, and a substance with an antipyretic effect called Lunbrofebrin is known. In addition, lumbrokinase and leth, substances that have thrombolytic activity are known, but they are not known at all for antiviral effects.

On the other hand, regarding plant viruses that have recently attracted attention, although pesticides exist as shown in Table 2, these pesticides have an inhibitory effect only on specific plant viruses, and have an inhibitory effect on a wide range of plants. There are general-purpose substances (agricultural chemicals) that can be brought about. [0009] [Table 2]

Pesticides against plant viruses

Product name Active ingredient Characteristics

Bis-Houguchin Tribasic Phosphate Degraded, inactivated, used for disinfection of equipment Lentamine Shiitake fungus cell extract Aggregates tobacco mosaic virus

Effective for soil-borne viral diseases mediated by DANICOL TPN filamentous fungus Patent Document 1: Patent No. 3273314

Patent Document 2: Japanese Patent Laid-Open No. 2003-259837

Disclosure of the invention

Problems to be solved by the invention

[0010] An object of the present invention is to provide a useful substance having an action of inhibiting virus infection and proliferation in a wide field including plants, livestock and fishery products in addition to plants. Means for solving the problem

[0011] The present inventors have focused on earthworms among organisms classified as lower organisms that do not have lymph, and have clarified their self-defense systems. As a result, it was found that earthworm-derived components have an effect of inhibiting infection and propagation of viruses against plant viruses. Furthermore, this earthworm-derived component includes not only viruses that infect plants but also influenza viruses. It has also been found that the virus that infects the animals mentioned above can prevent the virus from infecting the cells, and further inhibits the growth of the virus.

[0012] According to the present invention, there is provided a medicament having an action of inhibiting virus infection and proliferation, which comprises an active ingredient derived from earthworms. According to a preferred embodiment, the aforementioned medicament for use against mammals including humans, seafood or birds is provided.

Also, a pesticide that has an action of inhibiting the infection and growth of viruses that infect plants, a pesticide containing an active ingredient derived from earthworms, and a food or drink that has an action of inhibiting the infection and growth of viruses, which are derived from earthworms. The present invention provides a food / beverage product containing the active ingredient and a feed having an action of inhibiting the infection and proliferation of viruses, and containing the active ingredient derived from earthworms. [0013] In a preferred embodiment, an earthworm-derived active ingredient is provided having a molecular weight of 10,000 or more. The above-mentioned pharmaceuticals, agricultural chemicals, foods and drinks, and feeds, wherein the active ingredient derived from earthworms has an absorption characteristic at a wavelength of 280 nm; the above-mentioned pharmaceuticals, wherein the active ingredient derived from earthworms is an ingredient that degrades virus-coated proteins, Agrochemicals, food and drink, and feed are provided. In addition, it contains ingredients derived from earthworms and is used to prevent infection and proliferation of viruses. A feed with an indication that it is used for prevention is also provided by the present invention.

[0014] [Fig. 1] Gel filtration chromatography of earthworm-derived virus infection growth inhibitor of the present invention

It is a graph which shows the relationship between each density | concentration with the density | concentration of a protein by (Cefacryl S-200) and an infection growth inhibition rate, and each fraction.

FIG. 2 is a graph showing the relationship between the rate of infection inhibition by fractions I and II with the earthworm-derived viral infection growth inhibitor of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] Hereinafter, a method (crushing method) for preparing a sample earthworm-derived component (Sampnore solution) will be described.

1. Freeze dried earthworms, 50g, and crush them finely (before lyophilization, earthworms are fasted for 1 day).

2. Add 50 mM Tris-HCl (pH 7.0) buffer to the crushed material and suspend sufficiently. Water, a solvent containing water or a solvent may be used, but the pH is preferably 6 or less under acidic conditions.

3. Next, centrifuge (separation conditions; rotation speed '10, 000rpm, time '20min, temperature' 4 ° C)

Collect the supernatant.

4. This supernatant (sample solution) was used for virus infection growth inhibition experiments.

[0016] Further, a method for adjusting the Sampnore liquid from the earthworm by the electric shock method will be briefly described below.

1. Put 10-20 live earthworms in about 20ml of physiological saline (0.8% by weight).

2. Apply a voltage of 6 volts for about 10 seconds. Water, solvent containing water or solvent may be used, but voltage Is applied under acidic conditions, preferably pH 6 or lower.

As a result of 3., a yellow earthworm fluid (body cavity fluid) was obtained.

[0017] An experimental example in which the sample solution obtained above is used as a screening method for, for example, a plant virus infection inhibitor is described below.

1. Spread only the buffer solution with two cotton swabs on the two primary leaves of the grown leguminous plant cowpea (Vigna unguiculata). Spread the Sampnore liquid (supernatant obtained by the above-mentioned crushing method) on this leaf (another bud, Sampnore-treated leaf) using a cotton swab. If cowpea leaves repel the sample solution, mix 0.1% Triton (TritonX).

2. Then dry the primary cowpea leaves for 5 to 30 minutes.

3. Next, cucumber mosaic virus solution (CMV, p mark 0 strain, concentration 10 μg / ml to 20 zg / ml, 0.1 M phosphate buffer) and Carborundum 600 (scratches on cotyledon surface to 3%) Prepare a test solution mixed with

4. Soak two swabs in this test solution (virus solution) and take them out at the same time. Touch one of the swabs with the Sampunole treated leaf and the other swab with the control leaf and inoculate each leaf with a virus. .

5. Two to three days later, the number of local lesions of the virus was counted and the infection inhibition rate was examined.

[0018] Based on the above screening results, the results of the inhibitory action of earthworms on plant virus (cucumber mosaic wineles) infection are shown in the table below. The molecular weight fractionation of the sample was performed using an ultrafiltration module (Asahi Kasei Kogyo Co., Ltd.).

[Table 3] Sample Infection inhibition

1 Earthworm extract (extract with 50 mM Tris-HCl, pHL 0) 78.9%

2 Earthworm extract (molecular weight less than 10,000)-

3 Earthworm extract (Molecular weight 10,000 or more) 79. 7¾ In this table, infection inhibition% was calculated by the following formula.

Infection inhibition rate (%) = (1-number of treatment group / number of control group) x 100

That is, in sample 1 in the table, earthworm sample solution and kiyu on sample treated leaves The ratio of the number of virus lesions resulting from the mixture with the re-mosaic virus solution to the number of diseased lesions on the control leaves with only buffer solution, that is, the infection inhibition rate was 78.9%. As a result of molecular weight fractionation, the infection inhibition rate was 79.7% when the molecular weight was 10,000 or more, and the infection inhibition rate could not be clearly determined when the molecular weight was 10,000 or less.

This result was almost the same with the supernatant obtained by the electric shock method.

[0020] Next, a crude purification of the virus infection growth inhibitor from earthworms by gel filtration was attempted.

1. Freeze dried earthworm 50g and crush it finely (earthworm fasted 1 day before freeze-drying).

2. Add 50 mM Tris-HCl (pH 7.0) buffer to this crushed material and suspend well.

3. Centrifugation (separation conditions; number of revolutions • 10,000 ΓΡΠΙ, time '20 min, temperature '4 ° C), and collect the supernatant.

4. Add ammonium sulfate to this supernatant to 80% saturation and leave it overnight in a low greenhouse for salting out.

5. Further centrifugation (separation conditions; 10,000 mm, 20 min, 4 ° C), collect the precipitate, and dissolve in 20 mM Tris-HCl pH 7.0 buffer.

6. Perform gel filtration chromatography with S-acryl (S-mark hacryl) S-200 to collect the active fraction.

[0021] As a result of the above, as shown in Fig. 1, the vertical axis represents the protein concentration (A280 '280nm absorption characteristics) and the infection inhibition ratio (Inhibitory ratio)%, and the horizontal axis represents the fraction number. As a result, fractions No. 54 to 58 (referred to as fraction I) and fractions No. 59 to 64 were measured.

Peaks appeared (referred to as fraction II), and the amount of each protein determined was 188 μg / ml and 224 μg / ml.

In particular, at two peaks showing inhibitory activity, the infection inhibition rate was 80.2% for fraction No. 56 and 84.8% for fraction No. 60.

The molecular weight was estimated to be 40,000 for fraction No. 56 and 20,000 for fraction 60 from the results of gel filtration.

[0022] Next, No. 56 of fractions I and F. No. 60 of II (each referred to as a sample solution hereinafter) and virus were mixed with the above-mentioned fraction to obtain a test plant. Attempts were made to prevent infection by the method of inoculation. (material)

Virus: Tomato mosaic virus (ToMV), formerly called tobacco mosaic virus (TMV) tomato strain.

Test plants: tobacco leaves (Nicotiana tabacumu cv. Xanthin nc), tobacco with N gene introduced, show hypersensitive reaction to TMV (including ToMV) infection, and show local lesions (necrotic spots) at the site of infection . Ten test plants were prepared for a repeat number of 10.

[0023] (Experimental method)

1. Mix each of the two sample liquids (Νο · 56, Νο.60) and ToMV solution in an equal amount of 1: 1, respectively, and dilute the final concentration twice for each sample solution, and 50 µg / ml of ToMV solution. And

On the other hand, in the control group, a solvent (20 mM Tris-HCU 0.2M NaCl) and ToMV solution are mixed in an equal volume of 1: 1 and applied (the virus concentration is the same).

2. Vortex and mix.

3. Leave at 4 ° C for 30 minutes (temperature at which virus can stably exist, good on ice or in refrigerator).

4. Sprinkle carbon random on the maximum development of tobacco leaves grown in the 5-6 leaf stage.

5. Inoculate the sample solution and ToMV mixture, and the solvent and ToMV mixture, respectively, with the half leaf of the largest development leaf as the treatment group and the other as the control group.

6. Count the local lesions (necrotic spots) formed after 2-3 days and calculate the infection inhibition rate using the following formula.

Infection inhibition rate (%) = (1-number of lesions in treatment group / number of lesions in control group) X 100

[0024] As is clear from Fig. 2, the infection inhibition rate was about 58% in the sample solution of fraction Ι (Νο · 56) and about 85% in the sample solution of fraction II (Νο.60).

From the above results, it became clear that earthworm-derived active ingredients have antiviral properties, ie, virus infection inhibition and growth inhibition properties.

[0025] Next, a CMV inhibitory mechanism was analyzed for the virus infection growth inhibitory substance (fraction Π) from earthworms.

In order to determine the distribution of CMV-infected sites in the fraction II-treated leaves, a fraction of 400 zg / ml was applied to tobacco leaves and inoculated with CMV 30 minutes after complete drying. CMV inoculation 2, Four days later, the inoculated leaves were sampled and CMV-infected sites were detected by Tissue blot method.

As a result, the number of CMV-infected sites in the fraction II treated leaves was less than that in the control group 2 and 4 days after CMV inoculation. However, there was no difference between the infected site area in fraction II and the infected site area in the control group.

[0026] In order to investigate the possibility that Fraction II permeates into the treated leaves and inhibits CMV in the treated leaves, Fraction II 400 ag / ml was applied to the tobacco leaves on the back surface, and after 30 minutes of complete drying CMV was inoculated on the tobacco leaf surface. Four days after the inoculation, the inoculated leaves were sampled, and CMV-infected sites were detected by the Tissue biot method.

As a result, there was no difference in the number of CMV infection sites between fraction II and control.

[0027] CMV-coated protein (CMV CP) and fraction II are mixed and incubated at 30 ° C for 1 hour, then SDS-P

AGE was performed. After electrophoresis, the gel was collected and stained with CBB.

As a result, it was found that CMV CP was decomposed.

From the above results, it was clarified that Fraction II showed CMV CP resolution and could inhibit invasion into plants by direct contact with CMV.

When the N-terminal amino acid sequence was examined using an earthworm-derived plant virus infection inhibitor (fraction Π), it was SVGGSNAXPX-.

[0028] About the earthworm crush extract, Fraction I and Fraction II

The type A virus infection inhibitory effect was investigated.

The sample and human influenza A virus suspension were reacted at low temperature for 1 hour, then added to MDCK cells and infected for 1 hour. This reaction solution was removed, a cell culture solution was added, and the cells were cultured overnight. After fixing these cells, the number of infected cells was measured by immunostaining using antiviral monoclonal antibodies, and the infection inhibition rate was calculated. As a result, all the samples examined were stronger than Fetuin used for the positive control. Viral infection inhibitory activity was observed. In particular, the earthworm fragment extract and Fraction II were found to have very strong viral infection inhibitory activity.

[0029] The effect of inhibiting the red blood cell agglutination reaction of the earthworm crush extract was measured using Fetuin as a control. As a result, the same level of inhibitory activity was observed. Was found to have inhibitory activity.

[0030] In the above description, the power of the earthworm, the earthworm, etc. Any earthworm that produces an active ingredient having the same function can be used and is not limited.

In addition, it has been found that substances having a molecular weight of 10,000 or more and having absorption characteristics at 280 nm have a remarkable effect of inhibiting viral infection and growth.

Therefore, the active ingredient having an action of inhibiting the infection and propagation of the novel earthworm-derived virus of the present invention has an action of inhibiting the infection and propagation of the virus in a wide variety of species including plants, livestock and seafood. It is a substance that has the effect of sufficiently inhibiting the infection and proliferation of viruses even against SARS, koi herpes, and the like.

[0031] It is speculated that there is a common self-defense system for organisms that do not have lymph, and that the present invention is widely encompassed by organisms that do not have lymph that is not limited to earthworms. it is obvious.

[0032] Extracts, extracts, purified solutions or purified products obtained by extracting earthworms as described above from water, water-containing solvents or solvents can be used for foods and drinks or health foods. Health food means a food that is more active than ordinary food, and is intended for health, health maintenance and promotion. For example, the form is liquid or semi-solid, solid products, specifically powders, granules, tablets, capsules or liquids, and confectionery such as cookies, rice crackers, jellies, yokans, yognoreto, manju, etc. , Soft drinks, teas, nutritional drinks, soups, etc.

[0033] In the production process of these foods or to the final product, the above-mentioned active ingredients derived from earthworms can be added by mixing, coating or spraying to make a food or drink. When used as a food or drink, it is appropriate to use it in an amount that does not adversely affect the taste or appearance of the food or drink. For example, a solution containing 100 to 300 μg / ml of a protein having an action of inhibiting the infection and growth of the virus obtained after gel filtration chromatography as described above, in an amount of 0.01 to 2% by weight of the target food. You can get it.

[0034] Further, the above-mentioned earthworm is extracted with water, water-containing solvent or solvent power, and the extract, extract or purified solution or purified product thereof is feed for mammals, seafood, birds, etc. Alternatively, it can also prevent infection of animals with viruses by adding it to pet food. Earthworms for use as feed or pet food are dissolved in water and water. The amount of the extract, extract or purified solution or purified product obtained by extraction with the medium or solvent power is the same as that used in the above food and drink.

[0035] The above-mentioned earthworm is extracted with water, water-containing solvent or solvent power, and the extract, extract or purified solution or purified product thereof is water-based with or without mixing with an appropriate excipient. Alternatively, it can be used as an agrochemical in the form of an oily suspension, solution or emulsion. Examples of excipients that can be used include solvents (for example, oily solvents such as olive oil and soybean oil, hydrophilic solvents such as water, alcohol, and propylene glycol), emulsifiers (for example, surfactants), and suspending agents. (Eg, polyvinyl compounds, hydrophilic polymers such as cellulose, surfactants, etc.), bulking agents (eg, talc, clay, etc.), preservatives (eg, parabens, sorbic acid, etc.), colorants, etc. Can do.

[0036] The pesticide may be provided as a dry product that is regenerated with water or other suitable solution prior to use.

The agrochemicals described above can be applied by applying or spraying on plants.

For example, the agrochemical can contain 0.01 to 100 / g / ml of a protein having an action of inhibiting infection and growth of the virus obtained after the gel filtration chromatography.

Industrial applicability

[0037] According to the present invention, there are provided a pharmaceutical, a food, a drink, a feed or an agrochemical having an action of inhibiting viral infection and growth.

Claims

The scope of the claims

[1] A drug having an action of inhibiting virus infection and proliferation, which contains an effective ingredient derived from earthworms.

[2] The medicament according to claim 1, which is used for mammals including humans, seafood, or birds.

[3] An agrochemical that has an action of inhibiting the infection and growth of viruses that infect plants, and that contains an active ingredient derived from earthworms.

[4] A food / beverage product having an action of inhibiting the infection and proliferation of a virus and comprising an active ingredient derived from earthworms.

[5] A feed having an action of inhibiting virus infection and growth, and comprising an effective ingredient derived from earthworms.

[6] The earthworm-derived active ingredient is an ingredient having a molecular weight of 10,000 or more and / or an absorption characteristic at a wavelength of 280 nm, the medicine according to claim 1 or 2, the agrochemical according to claim 3, and the claim according to claim 4. Food or drink, or feed according to claim 5.

[7] The earthworm-derived active ingredient is an ingredient that degrades a viral coat protein or

The medicine according to claim 2, the agricultural chemical according to claim 3, the food or drink according to claim 4, or the feed according to claim 5.

[8] A food or drink product that contains a component derived from earthworms and is labeled as being used to prevent virus infection and proliferation.

[9] A feed containing an earthworm-derived component and labeled as being used to prevent infection and growth of viruses.