JP2009269861A - Prophylactic and therapeutic agent for viral infection derived from cassis fruit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a food, a medicine, a pet food and feed which are capable of preventing and treating viral infections without causing any adverse effects. <P>SOLUTION: The present invention is based on a finding that a cassis fruit extract contains a component effective in preventing infection of viruses, particularly influenza virus, and that the fruit extract does not cause any adverse effects by daily ingestion and is excellent in taste. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a virus infection preventive / therapeutic agent using an anti-influenza virus active ingredient contained in a cassis fruit and a food or drink or pet food or feed containing a cassis fruit extract.

  Influenza viruses are classified in the Orthomyxoviridae family and are classified into A, B and C types. Among them, influenza A virus is classified into a plurality of subtypes based on the difference in surface protein, and the antigenicity is easily mutated, so that influenza virus infection is prevalent every year. Infants with weak immunity and elderly people are terrible infectious diseases that can be caused by this influenza virus infection.

  However, effective and highly safe preventive and therapeutic agents for influenza viruses have not been developed yet. The reason is the various antigenic variations of influenza A virus.

  There are two types of genomic mutations that cause antigenic changes. One is called antigen shift and is characteristic of influenza virus. When two types of influenza virus infect the same host cell, mixed strains with different segments appear. Due to this antigen shift, there is a risk that influenza viruses that have hosted other organisms have the ability to infect humans, resulting in fatal diseases and the emergence of viruses that are pandemic worldwide. Infection of humans with the avian influenza virus currently attracting attention is caused by this antigen shift.

  Another antigenic mutation is a genomic mutation called antigen drift. Influenza A virus changes the amino acid sequence of the surface protein due to this genomic mutation, and viruses with different antigenicity appear even if they are the same subtype. Humans defend themselves by producing antigen-specific antibodies. However, specific antibodies lose their potency due to influenza virus mutations, causing epidemics every year.

  For prevention of influenza virus infection, vaccines are the mainstream. However, vaccines are produced by predicting the antigenicity of influenza virus based on the epidemic status of influenza and the immune status of healthy humans before the epidemic. However, as described above, influenza viruses are prone to antigen shift and drift, so vaccine antigens often do not match the prevalent viruses, and the preventive effects of vaccines are not satisfactory. Vaccination is not currently required.

  Furthermore, currently used vaccines are effective against A-Soviet, A-Hong Kong and B-types, but not against new viruses and avian influenza. In addition, amantadine hydrochloride and oseltamivir have been approved as influenza virus drugs, but side effects and the emergence of resistant viruses are feared.

  Japanese Patent Application Laid-Open No. 2002-145790 (Patent Document 1) discloses an anti-influenza virus agent containing as an active ingredient an extract of a rose family plant such as dog rose, apothecary rose, and Japanese Patent Application Laid-Open No. 11-130692. The gazette (Patent Document 2) discloses that the birch extract has antiviral activity against influenza viruses, but none of them is fully satisfied.

JP 2002-145790 A JP-A-11-130692 JP 2006-76954 A JP 2006-104121 A JP 2006-137712 A JP 2008-50301 A

  An object of the present invention is to provide foods, drugs, pet foods and feeds that have no side effects and can prevent and treat viral infections.

  As a result of intensive research, the present inventor has found that the cassis fruit extract contains a component having an effect of preventing infection of viruses, particularly influenza viruses. As a result, it was found that no side effects were caused even if it was taken in, and the taste was excellent, and the present invention was completed.

That is, the present invention
(1) An agent for preventing and treating viral infections comprising a cassis fruit extract as an active ingredient,
(2) Cassis fruit extract is mainly composed of fractions containing aromatic compounds (excluding Delphinidin-3-rutinoside, Delphinidin-3-glucoside, Cyanidin-3-rutinoside, Cyanidin-3-glucoside) (1) a viral infection preventive / therapeutic agent,
(3) The virus infection preventive / therapeutic agent according to (1), wherein the cassis fruit extract is an aromatic compound and contains a fraction eluted with 30% ethanol water by chromatography using an adsorption resin (HP20SS). ,
(4) A virus infection preventing / treating agent, wherein the virus according to (1), (2) or (3) is an influenza virus,
(5) A food / beverage product for the prevention / treatment of viral infection, comprising the agent for preventing / treating viral infection according to (1), (2), (3) or (4),
(6) The present invention relates to a virus infection prevention / treatment pet food or feed comprising the virus infection prevention / treatment agent according to (1), (2), (3) or (4).

Cassis ( Ribes nigrum L. ) is a plant belonging to the currant family, and its fruit is used for jelly, jam or liqueur. As a bioregulatory function of cassis, an antihypertensive action and an anti-herpesvirus action have been reported, and as an effect of cassis anthocyanin, an antioxidant action and an eyesight improving action have been reported.

  For example, Japanese Patent Application Laid-Open No. 2006-76954 (Patent Document 3) describes the use of cassis fruit as an anti-obesity agent and a blood cholesterol rise inhibitor, and Japanese Patent Application Laid-Open No. 2006-104121 (Patent Document 4). ), The use of cassis fruit as an intestinal agent is also proposed, and JP-A 2006-137712 (Patent Document 5) shows that cassis fruit is used as an antitumor agent, and JP-A-2008-50301. Japanese Patent Publication (Patent Document 6) describes using mate leaves, cassis fruits and the like as pancreatic lipase inhibitors.

  The cassis fruit extract of the present invention means that the fruit is neutral to acidic conditions (sulfuric acid, hydrochloric acid, acetic acid, etc.), water, organic solvents (alcohols, acetones, ethyl acetate, etc.), or a mixed solvent thereof. Extracted. Further, by removing organic acids or sugars from this extract with a synthetic resin or a reverse osmosis membrane, the effect of preventing virus infection can be enhanced, and a preferable one can be obtained. The cassis fruit used for extraction may be fresh, frozen or dried, and may be crushed. Furthermore, although the extraction method varies depending on the state of the raw material, the type of solvent used, etc., it is generally carried out under normal pressure from room temperature to heating conditions. For example, extraction using acidic water is preferably performed at room temperature (about 25 ° C.) for several minutes to several hours. In addition, the obtained extract may be concentrated by a vacuum concentrator or the like, removed from the solvent, spray dried, or converted into an aqueous solution and freeze-dried.

  Further, as the cassis fruit extract, a fraction containing an aromatic compound other than Delphinidin-3-rutinoside, Delphinidin-3-glucoside, Cyanidin-3-rutinoside, and Cyanidin-3-glucoside, which are anthocyanin compounds, is preferable. An appropriate purification treatment can be carried out to achieve a high content of the active ingredient, but there are no particular restrictions on the level of purification of the extract and the form of utilization. Such a fraction may be prepared by ultrafiltration, a permeable membrane, a synthetic resin, or the like. For example, after extracting frozen cassis fruit with 1.5% sulfuric acid solution, passing it through a synthetic adsorption resin, extracting with 60% water-containing ethanol, concentrating the fraction under reduced pressure and spray-drying, and then extracting the extract containing the fraction It only has to be obtained.

  When an influenza virus preventive / therapeutic agent containing a cassis fruit extract is used, it can be processed as it is or in various forms. In other words, various pharmaceutical substances acceptable for pharmaceuticals or foods, such as excipients, diluents, disintegrants, binders, coating agents, lubricants, lubricants, lubricants, flavors, sweeteners, emulsifiers, Solubilizers and the like can be included as adjuvants. Specific examples include titanium dioxide, lactose, mannitol, dextrin, dietary fiber, talc, gelatin, starch, cellulose and derivatives thereof, sugar, ascorbic acid, citric acid, polyethylene glycol, glycerin, animal and vegetable oils and the like. Examples of the form of the general food include fruit juice drinks, gums, strawberries, fermented milk, tea, carbonated drinks, jelly, pudding, gummy and the like. Moreover, as a pharmaceutical, it can be used as a mouthwash, a troche, a tablet, a granule, a powder, a powder, a capsule, etc.

  It is considered that the virus infection inhibitory effect of the present cassis fruit extract is mainly due to inhibition of virus adsorption to host cells and suppression of virus growth.

  Examples of viruses that can prevent infection with this extract include respiratory infection viruses such as influenza viruses, parainfluenza viruses, adenoviruses, coronaviruses, and digestive infection viruses such as rotaviruses. Among them, the activity against influenza viruses containing a sialic acid group in the host cell receptor is high, but the present invention is not limited thereto.

  By using a virus infection preventive / therapeutic agent and a food / beverage product for preventing or treating a virus infection comprising the cassis fruit extract as a main component according to the present invention, the virus infection can be prevented / treated with few side effects. Furthermore, it can be applied to pets and livestock by mixing with pet food and feed.

  Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Material preparation
"Preparation of Cassis fruit extract"
Add 2.0 kg of frozen cassis fruit and 6.0 L of 0.5% sulfuric acid water to a 10 L container, and stir at room temperature for 1 hour to extract. The extracted mixture is filtered off using a 10 mesh wire mesh. The solid residue was returned to a 10 L container, 5 L of 0.5% sulfuric acid was added, and the mixture was stirred for 30 minutes, and extracted and filtered in the same manner. The same operation was performed once more and extracted three times in total. The filtrate was mixed, 200 g of diatomaceous earth was added and stirred for 10 minutes, and then fine solids were filtered using filter paper to obtain about 16 L of an extract. This extract was passed through 700 ml of synthetic adsorption resin, and after passing through the resin, the resin was washed with 2 L of ion-exchanged water. Subsequently, the adsorbate was eluted using 3 L of 60% ethanol water. Under reduced pressure, the solvent was distilled off at a temperature of 50 ° C. or lower using an evaporator under reduced pressure to obtain 24.0 g of a cassis fruit extract.

  As a result of quantitative analysis by HPLC, the anthocyanin content was as follows: Del-3-R (Delphinidin-3-rutinoside) 19.0%, Del-3-G (Delphinidin-3-glucoside) 4.4 %, Cya-3-R (Cyanidin-3-rutinoside) 10.5% and Cya-3-G (Cyanidin-3-glucoside) 1.5%.

"Preparation of influenza virus solution"
The influenza virus strain used in the hemagglutination inhibition test was influenza virus A / Puerto rico / 8/34 (PR8 strain: H1N1). Culturing was carried out at 2 ° C. for 2 days and placed at 4 ° C. overnight, and allantoic fluid was collected. The influenza virus titer of this allantoic fluid was measured using MDCK cells (canine kidney-derived cells) and expressed as the reciprocal value of the dilution at 50% infection, which was 10 ^7.4 TCID ₅₀ / ml. This allantoic fluid was used as virus solution (PR8).

"Hemagglutination inhibition test method"
Influenza virus has the ability to adsorb chicken erythrocytes, which reflects the phenomenon of influenza virus adsorbing to respiratory mucosal epithelial cells. Therefore, a hemagglutination inhibition test was performed to determine whether each test substance inhibits the adsorption / aggregation process of the influenza virus to the target cells.

  50 μl of a solution prepared by adjusting each test sample to a concentration of 0.2 or 2.0 mg / ml with phosphate buffer (PBS) was mixed with 50 μl of PR8 and allowed to react on a 96-well round bottom plate at room temperature for 1 hour. After the reaction, the sample / virus mixture was diluted 2-fold on a plate with PBS containing 0.1% bovine serum albumin (BSA). Thereafter, 50 μl of chicken erythrocytes adjusted to 0.5% with 0.1% BSA-containing PBA were added to all wells and allowed to stand at room temperature for 1 hour, and then hemagglutination was confirmed. The highest dilution of virus causing complete hemagglutination (HA) was determined as the HA titer.

  The cassis fruit extract obtained by the above purification method was examined for the effect of suppressing hemagglutination. At this time, as a comparative objective, blueberry fruit extract (anthocyanidin content by absorbance method 25%), various anthocyanin standard products {Delphinidin-3-rutinoside (Del-3-R) and Delphinidin-3-glucoside (Del-3-G ) Were manufactured by Polyphenols, and Cyanidin-3-rutinoside (Cya-3-R) and Cyanidin-3-glucoside (Cya-3-G) were manufactured by EXTRASYNTHESE} and a mixture of each anthocyanin standard was similarly examined. Regarding the difference between the HA value control group and the test group, Student's t-test was performed using Statcel (OMS, statistical analysis software).

As a result, black currant extract 0mg / ml (control) and PR8 HA titer treated with 0.2 mg / ml were respectively 2 ¹⁰ and 2 ^9.3. Meanwhile In PR8 was treated with 2.0 mg / ml is 2 ^4.3, significantly higher hemagglutination inhibition inhibition those treated with 2.0 mg / ml was observed. Meanwhile, blueberry extract was HA titer 2 ^8.3 2.0 mg / ml (Figure 1). Further, when PR8 treated with cassis extract 2.0 mg / ml was 2 ^5.3, Delphinidin-3-rutinoside standard was 2 ^8.6 at a concentration of 2.0 ml / ml. Thus, although both blueberry and Delphinidin-3-rutinoside standard products showed inhibitory inhibitory action, the effect was low compared to the cassis extract (FIG. 2). As a result, it became clear that the cassis extract had a high hemagglutination-inhibiting action, which was not due to anthocyanins.

  A cassis fruit extract (5.03 g) was dissolved in 60 ml of deionized water, and 100 ml of a synthetic adsorbent was passed through a glass column. The column was washed with 300 ml of deionized water to obtain 0.50 g (yield 9.9%) of non-adsorbed material (F1). The column was washed three times with 200 ml of 15% ethanol water to obtain 1.91 g (yield 38.0%), F3 0.78 g (yield 15.5%), and F4 0.21 g (yield 4.2%). (FIGS. 3 and 4). Subsequently, it was washed three times with 200 ml of 30% ethanol water to obtain F5 0.08 g (yield 1.6%), F6 0.76 g (yield 15.1%) and F7 0.14 g (yield 2.8%). Further, it was washed with 1000 ml of 100% ethanol to obtain 0.30 g of F8 (yield 6.0%). As a result of quantitative analysis by HPLC, the anthocyanins contained in this fraction were F1: 0.0%, F2: 46.6%, F3: 57.6%, F4: 19.7%, F5: 9.34%, F6: 2.6%, F7: 0.4% and F8: 0.2%.

With each of these fractions at a concentration of 2.0 mg / ml, the above hemagglutination inhibition inhibition test was performed, the amount of components in each fraction was calculated based on the recovery rate by the above chromatographic purification, and the HA value was examined ( (Figure 5). While Control of HA was 2 ^11, F6 fraction showed a significantly higher inhibitory effect and 2 ^6.3. This was the same HA number as the 2.0 mg / ml cassis extract. From this, it was found that the active fraction was present in the F6 fraction. Furthermore, as a result of quantitative analysis by HPLC, this active fraction contains almost 2.6% of anthocyanins, and this also indicates that this high inhibitory action is not derived from cassis anthocyanins. all right.

  Using the above-mentioned cassis fruit extract and blueberry fruit extract, a test using influenza-infected model mice was conducted.

  BALB / c female mice (8 weeks old) were divided into 3 groups and 6 mice per group were used for the test. Cassis fruit extract and blueberry fruit extract were both adjusted to 0.1 and 0.2 mg / ml with PBS. PR8 diluted with PBS containing 0.1% BSA and test solutions of various concentrations were mixed at 37 ° C. for 3 minutes to obtain a reaction solution. 10 μl of each reaction solution (20 μl / mouse) was administered to both nasal cavities of mice anesthetized (65 mg / g body weight) by intraperitoneal administration of Nembutal to infect the lower respiratory tract. Thereafter, the state of onset and survival was observed for 14 days. Influenza development in mice was judged from weight loss, hair handstand, and slow movement. In the Control group, PR8 reacted with PBS instead of the test solution was infected with the lower respiratory tract.

  About the difference between the control group and the test group of the weight transition, Student's t test was performed using Statcel (OMS, statistical analysis software). Moreover, about the difference between the control group and test group of an incidence rate curve and a survival rate curve, the logrank test was performed using Statcel.

(body weight)
As a result of measuring the body weight over time, the control group started to decrease from the third day after the influenza virus infection, whereas the weight loss was not observed in the 0.2 mg / ml group treated with the cassis fruit extract. On the other hand, in the group treated with the cassis fruit extract 0.1 mg / ml, weight loss was observed from the 7th day after infection, but the decrease was significantly slower than that in the control group. However, in both blueberry fruit extract treatment groups, body weight decreased markedly after the fourth day after infection, and onset was observed (FIG. 6). From these results, it was suggested that components not contained in the blueberry fruit extract in the cassis fruit extract suppress influenza virus infection in a concentration-dependent manner.

(Incidence rate)
As a result of measuring the incidence of influenza over time, the control group reached 100% on the 5th day after infection, whereas the cassis fruit extract 0.2 mg / ml treatment group showed no onset and was significantly Suppressed. In addition, the cassis fruit extract 0.1 mg / ml treatment group showed an incidence of 50% in the observation period of 14 days, but was significantly suppressed compared to the control group. On the other hand, the blueberry fruit extract both treatment group developed 100% on the 6th day and could not reduce the onset rate (FIG. 7).

(Survival rate)
The survival rate of the control group began to be confirmed from the 7th day, and the survival rate was about 17% in the observation period of 14 days, whereas the cassis fruit extract 0.1 mg / ml and 0.2 mg / ml The treated group showed 100% survival rate and a significant increase in survival rate was observed. However, in the blueberry fruit extract treatment group, death gradually began to be confirmed after day 9, and the survival rate was about 33% in the 0.1 mg / ml treatment group and about 67 in the 0.2 mg / ml treatment group over the 14-day observation period. %, Which was significantly lower compared to the cassis fruit extract (FIG. 8).

The figure which shows the hemagglutination inhibition test result of a cassis fruit extract and a blueberry fruit extract. The figure which shows the hemagglutination inhibition test result of a cassis fruit extract and an anthocyanin reference standard. The figure which shows the fractionation method and recovery rate of a cassis fruit extract. The figure which shows the thin layer chromatogram of each fraction of a cassis fruit extract. The figure which shows the hemagglutination inhibitory effect in the separation fraction from a cassis fruit extract. The figure which shows the weight loss inhibitory effect in the influenza infectivity inhibitory effect of a cassis fruit extract. The figure which shows the onset rate suppression effect in the influenza infectivity inhibitory effect of a cassis fruit extract. The figure which shows the survival rate raise effect in the influenza infectivity inhibitory effect of a cassis fruit extract.

Claims (6)

  A virus infection preventive / therapeutic agent comprising cassis fruit extract as an active ingredient.   The cassis fruit extract is mainly composed of a fraction containing aromatic compounds (excluding Delphinidin-3-rutinoside, Delphinidin-3-glucoside, Cyanidin-3-rutinoside, and Cyanidin-3-glucoside). The preventive / therapeutic agent for viral infection according to claim 1.   The virus infection preventive / therapeutic agent according to claim 1, wherein the cassis fruit extract is an aromatic compound and contains a fraction eluted with 30% ethanol in water by chromatography using an adsorption resin (HP20SS).   A virus infection preventive or therapeutic agent, wherein the virus according to claim 1, 2 or 3 is an influenza virus.   A food / beverage product for the prevention / treatment of a viral infection comprising the agent for preventing / treating a viral infection according to claim 1, 2, 3 or 4.   A pet food or feed for virus infection prevention / treatment comprising the virus infection prevention / treatment agent according to claim 1, 2, 3 or 4.

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