JP2007223970A - Avian influenza virus infection inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a natural material-derived avian influenza virus infection inhibitor effectively inhibiting avian influenza virus infection. <P>SOLUTION: The avian influenza virus infection inhibitor comprises as active ingredient material(s) obtained from cacao beans, wherein the material is preferably at least one selected from cacao nib, cacao nib processed product, cacao husk and cacao husk extract. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an avian influenza virus infection inhibitor containing a substance obtained from cocoa beans as an active ingredient.

  Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family, and three types A, B, and C are known because of their antigenicity differences. Influenza virus has two types of glycoproteins, hemagglutinin (HA) and neuraminidase (NA) protruding in the form of spikes on the particle surface, and gene RNA segmented into eight segments is present inside. HA and NA on the surface of this virus are greatly mutated. For this reason, there are many types of influenza viruses, which are a cause of epidemics. Among them, influenza A virus has particularly many HA and NA mutations, so far, 15 major mutations have been found in HA and nine major mutations in NA, and there are as many subtypes as there are combinations thereof.

  In addition, subtype viruses that do not infect humans are considered to have birds and other mammals as hosts. In particular, all combinations of HA and NA have been found in waterfowl. Usually, these avian influenza viruses that use birds as a host are considered to have low infectivity from birds to humans, and there are few cases of human infection.

  However, the H5N1 type of avian influenza virus, a bird flu virus that has been devastating in recent years and that causes the death of the host, has been confirmed as a case of bird-to-human infection as of December 2005. Is growing.

  It is said that the infection route of influenza virus is mainly due to airborne infection. Influenza virus released by cough droplets enters through the nose and mouth, adsorbs to mucosal epithelial cells of the respiratory tract, and begins to grow after entering the cells. Because influenza viruses infect and proliferate in the airway mucosal epithelium and the epidemic type of the year cannot be accurately predicted, the effectiveness of vaccines varies greatly from year to year.

On the other hand, in recent years, studies have been made on the effect of suppressing the infection of influenza by natural materials, such as tea polyphenol component (see Patent Document 1), tea saponin (see Patent Document 2), and carrageenan-derived oligosaccharide (see Patent Document 3). , Rosaceae extract (see Patent Document 4), Raspberry, Strawberry, Blackberry, Fig, Akaza, Agrimony, Eucalyptus, Peach, Apple, Violet, Chromoji, Guarana, Cottonseed Rug, Tsuyuza, Nazuna, Enmeiso, Wild Strawberry , Hoahound, Marshmallow, Plantain, Lemon Verbena, Yarrow, Iceland Moss, Achacharu, Plant extract (see Patent Document 5), Orange, Cat's Claw, Bayberry, Lou, Everlasting, Linden, All Spa Scan, cocoa, plant extracts obtained from Karin that (Patent Document 6 reference) or the like is effective in mitigation of influenza have been reported.
Japanese Patent Laid-Open No. 03-101623 Japanese Patent Laid-Open No. 11-193242 Japanese Patent Laid-Open No. 2001-181188 JP 2002-145790 A JP 2004-059463 A JP 2005-343836 A

  In order for influenza virus to grow in living cells, the spike-like glycoprotein HA on the surface of the virus must first adsorb to a special place called a receptor on the surface of the cell. Then the virus cannot grow. For this reason, it seems that the reason that the avian influenza virus is difficult to infect humans is that the avian influenza virus cannot efficiently adsorb to human cells.

  As disclosed in Patent Documents 1 to 6, various influenza virus infection suppression effects by natural materials have been reported, but the influenza virus infection suppression effects by natural materials reported so far are mainly It is intended for human influenza viruses, and no infection-suppressing action against avian influenza virus has been reported, and a natural material having an action of suppressing avian influenza virus infection has not been particularly known so far.

  In addition, amantadine, a chemotherapeutic agent that is actually clinically applied to humans as an anti-influenza virus agent, is known to be effective against type A viruses but not to type B viruses. Yes. Thus, even if they belong to the same human influenza virus, the degree of effect varies greatly depending on the drug. Furthermore, it is thought that this is mainly due to the difference in antigenicity (difference in type) of HA required when influenza virus infects host cells. However, antibodies that can neutralize (inhibit infection) human influenza virus are not necessarily avian influenza. It is not always possible to neutralize viruses. That is, it is difficult to assume that materials and components having an infection inhibitory effect against human influenza virus have an infection inhibitory effect against avian influenza virus at the same time.

  Accordingly, an object of the present invention is to provide an avian influenza virus infection inhibitor derived from a natural material capable of effectively suppressing infection with an avian influenza virus.

  In order to complete the present invention, the present inventors have unexpectedly found that a substance obtained from cocoa beans has an effect of suppressing the growth of avian influenza virus while studying the physiological functions of components derived from cocoa beans. It came.

  That is, the avian influenza virus infection inhibitor of the present invention contains a substance obtained from cocoa beans as an active ingredient.

  In the present invention, the substance obtained from the cocoa beans is preferably at least one selected from cocoa nibs, processed cocoa nibs, cocoa husks, and cocoa husk extracts.

  In the present invention, the processed cocoa nibs are preferably at least one selected from cocoa mass, cocoa powder, and cocoa extract.

  In the present invention, cacao nibs and / or processed products thereof are contained in an amount of 0.01 to 100% by mass in terms of solid content, or cacao husks and / or extracts thereof are contained in an amount of 1 to 100% by mass in terms of solid content. It is preferable.

  The avian influenza virus infection inhibitor of the present invention can effectively suppress the growth of avian influenza virus.

  The avian influenza virus infection inhibitor of the present invention is a composition containing a substance obtained from cacao beans as an active ingredient, and preferably at least one selected from cacao nibs, processed cacao nibs, cacao husks, and cacao husk extracts It is a composition containing this.

  The substance obtained from the cocoa beans can be obtained, for example, as follows. That is, cocoa beans are taken out from cocoa fruits, and are usually fermented at about 30 ° C. for 4 to 7 days, and then dried. Then, after the dried cocoa beans are selected with a cleaner to remove foreign substances such as metals and stones, the cocoa nibs can be obtained by crushing with a separator and removing the skin and germs. At this time, the removed skin is cacao husk.

  The obtained cocoa nibs were added with an alkaline agent such as potassium carbonate, calcium carbonate, sodium carbonate, ammonium carbonate, ammonium bicarbonate, sodium bicarbonate, magnesium oxide, sodium hydroxide, or the like, if necessary, with respect to the weight of cocoa nibs. It may be immersed in a solution containing -5% by mass, preferably 2-4% by mass, and subjected to alkali treatment at 50-100 ° C. for 0-150 minutes, preferably 80-100 ° C. for 60-120 minutes. By this alkali treatment, the bitterness and astringency of cocoa beans can be removed.

  Moreover, as a cocoa nib processed material, the cocoa mass, cocoa powder, and cocoa extract which are generally used for chocolate and a cocoa drink can be used, for example, it can obtain as follows. That is, cacao mass can be obtained by roasting cacao nibs obtained as described above at 100 to 150 ° C. for 20 to 120 minutes and grinding with a grinder. Furthermore, cocoa powder (usually about 8-25% cocoa butter content) can be obtained by extracting the cocoa mass with a cocoa press to separate the cocoa butter and crushing it with a cocoa mill into a fine powder. . In the present invention, not only the cocoa powder (cocoa butter content 8% or more) specified in the “Fair Competition Rules for the Display of Chocolates”, but also the defatted cocoa (cocoa butter content 8) that cannot be said to be cocoa powder according to the above rules. %)) Can also be used. The cacao extract can be prepared by extracting cacao nibs, cacao mass and cocoa powder using, for example, a solvent such as water and alcohol, preferably a solvent such as hot water and ethanol. In the present invention, cocoa is particularly preferable as the processed cocoa nibs.

  Further, as described above, the cacao husk can be obtained as a by-product when collecting cacao nibs from cacao beans. Although cacao nibs are used as a chocolate raw material and a cocoa raw material, cacao husk has no particularly useful use, and most of them are currently disposed of.

  In the present invention, the cacao husk is preferably used after being roasted. The cocoa flavor can be improved at the same time as sterilizing by applying the roast treatment. The roasting treatment is preferably roasted at 70 ° C. or higher for 30 minutes or longer.

  When the cacao husk is used as it is, it can be used without being pulverized, or can be used after being pulverized so as to have a dozen μm depending on the application.

  The cocoa husk extract can be prepared by extracting the cocoa husk using, for example, a solvent such as water or alcohol, preferably a hot water or a solvent such as ethanol. Specifically, the cacao husk may be extracted in a solvent at 20 ° C. or higher for 15 minutes to 2 hours using the above solvent. In that case, it is preferable to carry out under pressure, for example, extracting under pressure of 1.1-3.0 atmospheres is preferable. For the extraction, any cacao husk crushed to an extent of about a dozen μm can be used, but those pulverized to 0.1 to 5 mm are preferred. When the cacao husk has a particle size of less than 0.1 mm, clogging is likely to occur during filtration after extraction, resulting in poor filtration efficiency, and exceeding 5 mm results in poor extraction efficiency. And after filtering the extract obtained in this way and distilling off the extraction solvent, the filtrate may be concentrated under reduced pressure and used as a concentrate, freeze-dried or spray-dried, and used as a dry powder. Good.

  The avian influenza virus infection inhibitor of the present invention is 0.01 to 100% by mass in terms of solid content of cacao nibs and / or processed products thereof, or 1 to 100% by mass in terms of solid content of cacao husks and / or extracts thereof. It is preferable to include. And in the avian influenza virus infection inhibitor of this invention, when administering to a human as a substance obtained from the said cocoa bean, using cocoa nib and / or its processed material is administered to poultry and livestock. In this case, the use of cacao husk and / or cacao husk extract is desirable from the viewpoint of effective utilization of what has been treated as an unnecessary material in the past, but can be appropriately selected depending on the usage form, usage method, and the like. In the present invention, cacao nibs, processed cacao nibs, cacao husks, and cacao husk extracts can also be used as the present avian influenza virus infection inhibitors as they are.

  Moreover, the avian influenza virus infection inhibitor of this invention can contain saccharide | sugar, a vitamin, minerals, an excipient | filler, dairy products, a fragrance | flavor, etc. other than said basic component.

  The avian influenza virus infection inhibitor of the present invention is used in various fields such as pharmaceuticals and foods, and can be used as active pharmaceutical ingredients, food raw materials and the like.

  For example, in the field of pharmaceuticals, the avian influenza virus infection inhibitor of the present invention can be formulated with a pharmaceutically acceptable substrate or carrier and provided as a pharmaceutical composition. This pharmaceutical composition includes, in addition to a base material and a carrier, a binder, a disintegrant, a buffer, a preservative, a moisturizer, an antibacterial agent, a preservative, a fragrance, a pigment, You may mix | blend additives, such as surfactant, a stabilizer, a solubilizing agent, arbitrarily. And as a form of the said pharmaceutical composition, either an internal form or an external form may be sufficient. For example, as a pharmaceutical composition administered in an internal form, dosage forms such as pills, powders, tablets, granules, capsules, syrups, solutions, jellies, lozenges and the like can be exemplified. Examples of the pharmaceutical composition administered in an external form include dosage forms such as ointments, solutions, emulsions, suspensions, patches, sprays, and sprays.

When the avian influenza virus infection inhibitor of the present invention is used as a pharmaceutical composition, the effective dose is 50 to 500 mg / kg body weight per day in terms of cacao nibs, preferably 100 to 300 mg / kg. The weight is kg or 200 to 5000 mg / kg of body weight per day in terms of cacao husk, preferably 500 to 3000 mg / kg of body weight. When used as pharmaceutical composition for external use is daily 0.01 - 100 / cm ² in nibs terms, or preferably 0.1 to 10 mg / cm ^2, per day in Kakaohasuku terms 0.1 a 1000 mg / cm ^2, preferably from 1 to 100 mg / cm ^2.

  When the avian influenza virus infection inhibitor of the present invention is used as a pharmaceutical composition, the blending ratio of the avian influenza infection inhibitor of the present invention in the pharmaceutical composition is the effective amount, the dosage form of the pharmaceutical composition, and the administration form. It is set according to etc.

  For example, in the case of a pharmaceutical composition to be administered in an internal form, 0.01 to 100% by mass, preferably 0.1 to 100% by mass, more preferably 0.1% by mass in terms of cacao nib relative to the total amount of the pharmaceutical composition. Is 1 to 50% by mass, or 0.1 to 100% by mass, preferably 1 to 100% by mass, and more preferably 10 to 100% by mass in terms of cacao husk. In the case of a pharmaceutical composition administered in an external form, 0.01 to 100% by mass, preferably 0.1 to 100% by mass, more preferably 1 in terms of cacao nib, based on the total amount of the pharmaceutical composition. The ratio which becomes -50 mass%, or 0.1-100 mass% in conversion of cacao husk, Preferably it is 1-100 mass%, More preferably, the ratio becomes 10-100 mass%. In addition, when using the avian influenza infection inhibitor of this invention as a pharmaceutical composition, it is preferable to use a cocoa nib and / or its processed material as a substance obtained from a cocoa bean.

  In the field of foods and drinks, the avian influenza virus infection inhibitor of the present invention of the present invention can be used in combination with foods for specified health use, dietary supplements, functional foods, etc. in addition to general foods. Examples of such foods include confectionery such as chocolate, biscuits, gum, candy, cookies, gummies, and tableted confectionery; cereals; beverages such as powdered drinks, soft drinks, milk drinks, nutrition drinks, carbonated drinks; ice Examples include frozen desserts such as cream and sherbet. Moreover, in the case of foods for specified health use, dietary supplements, and the like, they may be in the form of powders, granules, capsules, syrups, tablets, sugar-coated tablets and the like.

  When used in the field of foods and drinks, the blending ratio of the avian influenza virus infection inhibitor of the present invention is appropriately set according to the effective amount, food form and the like. As an example, it is 0.1 to 100% by mass in terms of cacao nibs, preferably 1 to 50% by mass, more preferably 3 to 50% by mass, or 1 to 100% in terms of cacao husk based on the total amount of food. %, Preferably 10 to 100% by mass. In addition, when using the avian influenza infection inhibitor of this invention in the field | area of food-drinks, it is preferable to use a cocoa nib and / or its processed material as a substance obtained from a cocoa bean.

  Moreover, the avian influenza virus infection inhibitor of this invention can be utilized besides the use to a pharmaceutical and food-drinks, for example, can also be used as filters, such as a poultry and livestock feed, a flooring, a mask. When the avian influenza infection inhibitor of the present invention is used in the field of poultry and livestock feed, bedding, filters such as masks, etc., cacao husk and / or extract thereof are used as substances obtained from cacao beans. It is preferable to use it.

[Preparation of cocoa extract]
The extract was extracted from the cocoa using hot water as a solvent to the cocoa pulverized and adjusted to 5 to 500 μm to obtain a 5% cocoa extract.
[Preparation of cacao husk extract]
An extract was extracted from the cacao husk into hot cacao husk as a solvent in a cacao husk pulverized to 10 to 50 μm to obtain a cacao husk extract having a concentration of 5%.

(Test Example 1)
About the anti-avian influenza virus effect by the said cocoa extract and the said cacao husk extract, the inhibition rate was measured and evaluated by FFU assay (Focus Forming Unit Assay). H5N1 avian influenza virus (A / Kyoto / 04) was used as the target virus.

  First, cocoa extract and cacao husk extract (test sample) are diluted 5 times with MEM medium in a 96-well microplate (2%, 0.4%, 0.08%, 0.016%). , 0.0032%) was added. Next, about 200 FFU / 50 μl MEM of avian influenza virus was added, mixed and allowed to stand for 30 minutes. Then, the above mixed solution was added to MDCK cells cultured in a 96-well microplate to infect the avian influenza virus and cultured for 16 hours. Thereafter, the cells were fixed with ethanol, and infected cells (1 FFU = 1 infectious virus) were stained by an enzyme antibody method using an anti-A-type NP (nucleoprotein) monoclonal antibody, the number of infected cells was counted, and the following formula (A ), The infection rate (%) of avian influenza virus was measured. The result of the avian influenza virus infection rate by the test sample of each concentration is shown in FIGS.

  From the results shown in FIG. 1, cocoa has an action to inhibit infection against avian influenza virus, and the infection rate of avian influenza virus can be reduced to 15% or less with a cocoa extract at a concentration of 0.008%. It was.

  Moreover, the cacao husk has the effect | action which inhibits infection with respect to avian influenza virus, and the infection rate of the avian influenza virus could be reduced to 10% or less with the cacao husk extract of 1% concentration.

(Test Example 2)
The cytotoxicity of the cocoa extract and the cacao husk extract was evaluated by the MTT method, which is a method for evaluating cell viability by measuring the activity of the enzyme dehydrogenase present in the mitochondria of the cells.

  MDCK cells were allowed to act on a 5-fold serial dilution (2%, 0.4%, 0.08%, 0.016%, 0.0032%) of cocoa extract and cacao husk extract, and cultured for 16 hours. MMT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide), a substrate for dehydrogenase, was added, and after 3 hours of reaction, the cells were lysed with a detergent and the substrate turned purple Absorbance was measured with a colorimetric spectrophotometer, and cytotoxicity was calculated from the following formula (B). The results are shown in FIGS.

  From FIG. 3, when the cocoa extract was added to the culture solution while culturing MDCK cells for 16 hours, no cytotoxicity was observed at a concentration of 0.2% or less.

  In addition, from FIG. 4, when the cacao husk extract was added to the culture solution while MDCK cells were cultured for 16 hours, no cytotoxicity was observed at a concentration of 2% or less.

It is a graph which shows the infection rate of the avian influenza virus by a cocoa extract. It is a graph which shows the infection rate of the avian influenza virus by a cacao husk extract. It is a graph which shows the cytotoxicity by a cocoa extract. It is a graph which shows the cytotoxicity by a cacao husk extract.

Claims (5)

  An avian influenza virus infection inhibitor comprising a substance obtained from cocoa beans as an active ingredient.   The avian influenza virus infection inhibitor according to claim 1, wherein the substance obtained from the cocoa beans is at least one selected from cocoa nibs, processed cocoa nibs, cocoa husks, and cocoa husk extracts.   The avian influenza virus infection inhibitor according to claim 2, wherein the processed cocoa nibs is at least one selected from cocoa mass, cocoa powder, and cocoa extract.   The avian influenza virus infection inhibitor of Claim 2 or 3 which contains 0.01-100 mass% of cacao nibs and / or its processed material in conversion of solid content.   The avian influenza virus infection inhibitor of Claim 2 which contains the said cacao husk and / or its extract 1-100 mass% in conversion of solid content.