JP2009149552A - Preventing agent and treating agent for hepatitis c virus infection disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material effective for suppressing the production of hepatitis C virus and pharmaceuticals and foods produced by using the material. <P>SOLUTION: A processed product of the tuber of a plant of the family Araceae has a specific action to suppress the production of hepatitis C virus. The tuber of the plant of the family Araceae itself may be used as the processed product, however, it is preferable to properly select and use from a dry crushed product, squeezed juice and extract according to the use. The use of a processed skin product among the tubers of the plant of the family Araceae is most suitable from the viewpoint of getting in high efficiency and activity. The HCV production inhibitor is applied to an HCV infected person for the prevention and/or treatment of diseases induced by HCV, for example, the onset of hepatitis of the HCV infected person, hepatitis of acute phase and chronic phase, the onset of hepatocirrhosis and hepatic cancer from chronic hepatitis or diseases aggravated by the virus infection diseases. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition for preventing and treating hepatitis C virus infection.

  The present invention also relates to a food composition for preventing and treating hepatitis C virus infection.

  The number of people infected with hepatitis C virus (HCV) is estimated to be 2 million in Japan, 5 million in the United States, and 170 million worldwide. Epidemiological survey results are revealing the route of HCV infection. However, the number of HCV infections is still increasing. HCV-infected persons continuously inflame and eventually progress to chronic hepatitis, cirrhosis, and liver cancer. It is also known that even if a cancerous part is removed by surgery, there are many patients whose liver cancer recurs due to inflammation that continues in the noncancerous part.

At present, interferon treatment is performed as the only treatment method for eliminating HCV, but about 30% of HCV infected patients are effective. HCV treatment is also being performed in combination with the antiviral drug ribavirin, but the response rate is only slightly higher than 30%, the effectiveness rate is still low, and a drug that is still effective for all HCV-infected patients has been found It has not been. In addition, interferon treatment has strong side effects such as hair loss, loss of appetite, depression, and platelet reduction, and there is also a problem with long-term continuous treatment (Non-patent Document 1). Therefore, in this technical field, development of an HCV treatment method and a therapeutic agent that have few side effects and can be taken for a long time is eagerly desired.

  By the way, nonpatent literature 2 can be mention | raise | lifted as literature which discloses the pharmacological function paying attention to the component derived from a taro similarly to this invention. Non-patent document 2 describes that a methanol extract of the asteraceae of the Araceae family has an antiviral action against bovine herpesvirus (Bovine herpesvirus type 1), but does not mention the effect on HCV. . However, as disclosed in Non-Patent Document 3, herpes virus and HCV are different viruses, and it cannot be said that a substance having a herpes virus production inhibitory action has a HCV production inhibitory action. In addition, as in the present invention, Patent Document 1 can be cited as a document disclosing its pharmacological function by focusing on components derived from natural products. Patent Document 1 discloses that plant components derived from blueberry leaves have an HCV production inhibitory action. However, blueberry leaves and taro are distantly related from the standpoint of plant taxonomy, and Patent Document 1 does not disclose or suggest that taro has an HCV production inhibitory action.

JP2007-119398A “Recent Topics on Chronic Hepatitis C Side Effects of IFN and Countermeasures” Med Dig Vol. 46, no. 6, 19-22 (1997.11) A. R. McCutcheon et al “Antivital screening of British Columbian Plants” Journal of Ethanolpharmacology, 49, 101-110 (1995). Nagata Keisuke, “Biology of Viruses-Central Dogma”, Yodosha Publishing, 1996

  An object of the present invention is to provide an HCV production inhibitor that is derived from a tuber of a taro family plant that has been regularly used as a food since ancient times, has few side effects, can be taken for a long period of time, and has an excellent effect, and a pharmaceutical containing the HCV production inhibitor It is to provide a composition.

  The subject of the present invention also includes an HCV production-suppressing material and an HCV production-suppressing material that are derived from tubers of taro family plants that have been regularly used as food since ancient times, have few side effects, can be taken for a long period of time, and are excellent in effect. It is to provide a food composition.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that tubers of taroaceae plants have an HCV production inhibitory action, leading to the present invention.

The present invention that has achieved the above object is characterized by the following aspects.
Item 1: A hepatitis C virus production inhibitor comprising a processed tuber of a taro plant as an active ingredient.
Item 2: The hepatitis C virus production suppression according to claim 1, wherein the processed product of the taro family plant is at least one selected from the group consisting of a crushed tuber, a squeezed tuber, and a tuber extract. Agent.
Item 3: The hepatitis C virus production inhibitor according to claim 1 or 2, wherein the processed product of the taro family plant is a tuber skin.
Item 4: A pharmaceutical composition comprising the hepatitis C virus production inhibitor according to claim 1, which is used for suppressing virus production for a person infected with hepatitis C virus.
Item 5: The method according to claim 1, which is used for preventing and / or treating a disease induced by hepatitis C virus or a disease aggravated by these virus infections for a person infected with hepatitis C virus. A pharmaceutical composition comprising a hepatitis C virus production inhibitor.
Item 6: A pharmaceutical composition comprising a hepatitis C virus production inhibitor according to claim 1, which is used to suppress the onset of hepatitis induced by hepatitis C virus for persons infected with hepatitis C virus.
Item 7: A hepatitis C virus production suppression material comprising a processed product of tubers of Araceae plants as an active ingredient.
Item 8: The hepatitis C virus production suppression according to claim 8, wherein the processed product of the taro family plant is at least one selected from the group consisting of a crushed tuber, a squeezed tuber, and a tuber extract. material.
Item 9: The hepatitis C virus production-suppressing material according to claim 8 or 9, wherein the processed product of the taro family plant is a tuber skin.
Item 10: A food composition comprising the hepatitis C virus production-suppressing material according to claim 7, which is used to suppress virus production for a person infected with hepatitis C virus.
Item 11: The method according to claim 7, which is used for preventing and / or treating a disease induced by hepatitis C virus or a disease aggravated by these virus infections for a person infected with hepatitis C virus. A food composition comprising a hepatitis C virus production-suppressing material.
Item 12: The food composition containing the hepatitis C virus production-suppressing material according to claim 7, which is used to suppress the onset of hepatitis induced by the hepatitis C virus for a person infected with hepatitis C virus.

  In addition, in this invention, a tuber shows the whole underground part of the stem of a taro family plant.

  An HCV production inhibitor and / or an HCV production inhibitor material comprising as an active ingredient a processed product of a tuber of a taro family plant of the present invention, for example, a pulverized product of a taro plant tuber, a juice of a tuber, and an extract of a tuber. HCV-induced diseases, such as the onset of hepatitis, acute and chronic hepatitis, and chronic hepatitis to cirrhosis and liver cancer, or diseases that are exacerbated by viral infection Suitable for prevention and / or treatment.

  In addition, since the tuber of a naturally derived taro plant that can be originally used for food is used as an active ingredient, there are few side effects and it can be safely taken or ingested over a long period of time. Especially for patients who are difficult to administer interferon, there is no suitable alternative treatment so far, which is a great gospel.

  The taroaceae plant used in the present invention is a cultivated plant native to Southeast Asia belonging to the genus Araceae. Tubers that are the underground part of the stem are edible. The petiole is also edible. Konjac (Amorphophallus rivieri var. Konjac) is also processed into food. There are several genera for ornamental purposes. In the present invention, the type, origin, and place of origin of the taro to be used are not particularly limited. Mucin, galactan, and potassium are known as components contained in tubers of Araceae plants. Mucin is known to prevent mucosal damage and prevent gastrointestinal wall ulcers. Galactan is known to have effects such as activation of brain cells and enhancement of immunity. Potassium is said to excrete sodium and prevent hypertension. However, there has been no report on the function of inhibiting HCV production by tubers of Araceae plants so far.

  The tubers of Araceae plants are processed before use. As a processing method, a method of pulverizing tubers as they are can be used, but dry pulverization of tubers, juice extraction of tubers, and solvent extraction of tubers are practical. In order to effectively exhibit the HCV production inhibitory action, it is preferable to use a treated product of tuber skin.

  Examples of the method for preparing the dried pulverized product include a method in which the tuber is dried as it is and then pulverized or cut into small pieces and then dried. The drying method is not particularly limited as long as the pharmacological effect of the present invention is not impaired, and vacuum freeze drying, hot air drying, far-infrared drying, vacuum drying, microwave vacuum drying, superheated steam drying, and the like can be widely used. Of these, it is practical to use vacuum freeze-drying with little component change. The vacuum freeze-drying conditions cannot be specified because they vary depending on the state of the raw material. For example, when the tubers are dried as they are, the freezing temperature is −30 ° C. to −20 ° C., the drying temperature is −30 to 30 ° C., and the drying time is 15 hours to A range of 24 hours is desirable. In the case of warm drying, the temperature is preferably 30 ° C. to 50 ° C. and the drying time is preferably in the range of 15 hours to 24 hours.

  In the preparation of the solvent extract, the tuber is extracted as it is or after being crushed, or is dried and then pulverized and extracted as necessary. Juice obtained by squeezing tubers can also be used as an extraction raw material. In this case, the stock solution may be concentrated or dried as necessary.

  The extraction solvent to be used is not particularly limited, but it is preferable to use water or a polar solvent compatible with water. Examples of polar solvents compatible with water include lower alkyl alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, and butanol; glycols such as ethylene glycol, butylene glycol, propylene glycol, and glycerin; ethyl ether, acetone, and the like Can also be used. From the viewpoint of safety, it is practical to use a lower alcohol, particularly ethanol, as the sole solvent.

  As the mixed solvent, water and the polar solvent may be used in combination, or two or more of the polar solvents may be used in combination. As an example of the latter, it is possible to use, for example, a mixed solvent of acetone and ethyl ether, preferably a 1: 1 (v / v) mixed solution of acetone and ethyl ether. In general, it is desirable to use a mixed solvent (hydrous solvent) of the above polar solvent and water. The hydrous solvent is hydrous alkyl alcohol, more preferably hydrous ethanol. The alcohol concentration in the hydrous alcohol is generally 5 to 90% by volume, preferably 30 to 90% by volume, and more preferably 50 to 90% by volume.

  As an extraction method, it is practical to immerse the raw tuber as it is in a crude powder, shredded product, or a dry pulverized product thereof in a solvent by cold immersion, digestion or the like. Extraction can be performed by stirring under heating and filtering. Alternatively, a percolation method may be used. For example, in stirring extraction with an 80% by volume ethanol aqueous solution, the solution temperature is desirably room temperature, and the immersion time varies depending on the temperature, but is preferably in the range of 30 seconds to 1 hour.

  From the obtained extract, solids are removed by filtration or centrifugation as necessary. The obtained filtrate may be used as it is or may be used after partially concentrating or drying by distilling off the solvent, depending on the next step. After concentration or drying, it can be used after washing and purifying with an appropriate washing solvent such as a non-soluble solvent, or it can be further dissolved or suspended in an appropriate solvent. In the present invention, the solvent extract obtained as described above can be used as a dried tuber extract by drying under reduced pressure, freeze drying or the like.

  The processed product of the taro family tuber thus obtained has a remarkable HCV production inhibitory action in the edible part and the skin, which are tubers of the taro family plant, as shown in Experimental Example 2 described later. Yes. Specifically, as can be seen from Table 1, in the taro edible tuber edible portion methanol-extracted sample, the 50% viable cell number concentration is 100 μg / ml or more in all varieties, and the 50% luciferase activity concentration is 3 It was 25 to 63.37 μg / ml. Also, in the taro plant tuber bark methanol extraction sample, as can be seen from Table 2, the 50% viable cell number concentration is 25.26 to 133.01 μg / ml, and the 50% luciferase activity concentration is 1.72 to 3. It was 62 μg / ml. As can be seen from the results, the taro plant tuber has the effect of suppressing the production of HCV replicon RNA without affecting the cells, and has the same effect as that of interferon shown in FIG. The HCV production inhibitor of the invention suppresses HCV RNA production, thereby causing a disease induced by HCV, such as hepatitis onset in HCV-infected persons, acute and chronic hepatitis, and chronic hepatitis to cirrhosis and liver cancer, or It exhibits the effect of preventing and / or treating diseases that are exacerbated by viral infections.

  The agent for inhibiting HCV production of the present invention can be used as a pulverized tuber, juice or extract itself in a single solid or liquid form, but if necessary, a pharmaceutically or food acceptable carrier Or an additive can be mix | blended and it can also provide as a solid or liquid pharmaceutical or functional food or drink. Also, other antiviral agents can be blended as active ingredients.

  When the HCV production inhibitor of the present invention is used as a pharmaceutical, its form is not particularly limited, but is preferably a form suitable for oral administration. Solid pharmaceutical preparations for oral administration are in the form of tablets, pills, capsules, fine granules, granules, etc., and liquid pharmaceutical preparations for oral administration are emulsions, solutions, suspensions, syrups. The form of an agent etc. can be mentioned. In these preparations, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusters and the like according to various preparations can be appropriately blended. In addition, as the main use of the pharmaceutical of this invention, the hepatitis therapeutic agent which can be used effectively for HCV production inhibitor or chronic hepatitis prevention, liver cirrhosis onset prevention, or liver cancer onset prevention can be mentioned.

  The amount of the active ingredient contained in a pharmaceutical product varies depending on the formulation form, the type of disease to be applied, and the like, and cannot be generally defined, but may be set as appropriate according to the dose per administration. The administration time is not limited, but is preferably the acute phase, chronic phase, or cirrhosis of hepatitis.

  Even when the HCV production-suppressing material of the present invention is used as a functional food or drink, its form is not particularly limited. For example, tablets, pills, capsules, granules, powders, powders, lozenges, or solutions (drinks) with the above-mentioned HCV production-suppressing material, together with carriers and additives that can be blended on food as needed Etc. can be prepared.

As an application example of functional foods and drinks, the following forms added with the HCV production-suppressing material of the present invention can be mentioned.
(1) Milk beverage, lactic acid bacteria beverage, soft drink with fruit juice, soft drink, carbonated drink, fruit juice drink, vegetable drink, vegetable / fruit drink, alcoholic drink, powdered drink, coffee drink, tea drink, green tea drink, barley tea drink, etc. Beverages.
(2) Puddings such as custard pudding, milk pudding, souffle pudding, pudding with fruit juice, desserts such as jelly, bavaroa and yogurt.
(3) Ice cream, ice milk, lact ice, milk ice cream, ice cream and soft ice cream with fruit juice, ice candy, sherbet, frozen confectionery such as ice confectionery.
(4) Plate-like or sugar-coated tablet gums such as chewing gum and bubble gum.
(5) Chocolates such as chocolate to which flavors such as strawberry chocolate, blueberry chocolate and melon chocolate are added in addition to coating chocolate such as marble chocolate.
(6) Hard candy (including bonbon, butterball, marble, etc.), soft candy (including caramel, nougat, gummy candy, marshmallow, etc.), caramels such as drop, toffee, etc. (7) Hard biscuits, cookies, rice cakes, Baked confectionery such as rice crackers (8) Consomme soup, soups such as potage soup (9) Various seasonings such as miso, soy sauce, dressing, ketchup, sauce, sauce, sprinkles (10) Strawberry jam, blueberry jam, marmalade, Jams such as apple jam, apricot jam, presabu.
(11) Fruit wine such as red wine.
(12) Processed fruits such as syrup pickled cherries, apricots, apples, strawberries and peaches.
(13) Processed meat products such as ham, sausage and grilled pork.
(14) Fish pasteurized products such as fish ham, fish sausage, fish surimi, salmon, bamboo rings, hanpen, fried Satsuma, Date roll, whale bacon.
(15) Noodles such as udon, cold wheat, somen, buckwheat, Chinese noodles, spaghetti, macaroni, rice noodles, harsame and wonton.
(16) Various other prepared foods and various processed foods such as rice cakes and rice fields.

  The content and intake of the HCV production-suppressing material of the present invention in functional foods and drinks are not particularly limited. Although it can select suitably according to the kind of food and drink, the target function, efficacy, and other various conditions, a desirable intake is an amount effective for HCV production suppression. Moreover, although the intake time is not limited, it is preferably the time before the onset of hepatitis, the acute phase of chronic hepatitis, the timing of cirrhosis.

  The functional food / drink of the present invention has an HCV production inhibitory effect, a hepatitis onset inhibitory effect, a hepatitis progression inhibitory effect, a cirrhosis onset preventive effect, or a liver cancer onset preventive effect based on containing an HCV production inhibitory material. . For this reason, the functional food and drink of the present invention is a food and drink intended for HCV-infected persons. For example, the functional food or drink is preferably taken before the onset of hepatitis, in the acute or chronic phase of hepatitis, or at the time of cirrhosis. Food and drink that can be done.

  The preparation and test method of replicon cells used in the following tests were as follows. In the examples, “%” means “W / W%” unless otherwise specified.

  Preparation of replicon cells: Genomic RNA of HCV is roughly classified into a core and envelope structural protein translation region constituting a virus particle, and a nonstructural protein translation region functioning for viral genome replication. A subgenomic replicon RNA was prepared by substituting this structural protein translation region with a luciferase translation region, EMCV IRES (brain myocardial virus internal ribosome binding sequence), or a neomycin resistance gene, and the obtained RNA was used in human hepatoma cell Huh-7. When introduced into the cytoplasm, Huh-7, into which the subgenomic replicon RNA has been introduced, has neomycin resistance at the same time, allowing selection with neomycin. The cells thus obtained were used for evaluation of the production amount of HCV replicon RNA. The amount of HCV replicon RNA produced was measured by the luciferase assay method described below. The cells were cultured using DMEM [GIBCO GlutaMAX Media Dulbecco's Modified Eagle Medium (D-MEM) (1 ×), liquid (High glucose, continents sodiumHybne)] -A medium supplemented with 1% Streptomycin (GIBCO) and 1% Genticin (Invitrogen) is used. The assay medium used for the assay was DMEM with 5% FBS and 1% Penicillin-Streptomycin (however, Geneticin was not added).

  Luciferase assay: Luciferase catalyzes the reaction of making luciferin oxide and AMP from luciferin and ATP in the presence of magnesium. The luciferase assay method is a method in which light generated at this time is detected by a luminescence detector, and luciferase activity is evaluated based on the obtained light quantity. In the present invention, for the sake of convenience, this light amount is defined as the amount of HCV replicon RNA.

  WST-8 assay: The novel tetrazolium salt WST-8 is reduced by intracellular dehydrogenase to produce water-soluble formazan. Since the amount of formazan produced is proportional to the number of living cells, in the present invention, the number of living cells was evaluated from the absorbance of formazan, and the cytotoxic effect of the test sample was determined.

<Experimental example 1>
(1) Preparation of methanol extraction sample of taro Sennan Nakano Satsumo Ichinaka Nakao's skin and edible portion were vacuum freeze dried (vacuum freeze dryer, vacuum freeze dryer, FTS SYSTEM, Dura-Top MP & Dura-Dry MP). Subsequently, it was pulverized through a 0.5 mm screen of an ultracentrifugal crusher (MRK & RETSCH, EM-1 type) to obtain a dried powder product of each skin and edible part of Satoimo Sennan Nakano. To about 3 g of the lyophilized powder of taro obtained, about 100 ml of methanol was added and shaken at room temperature for 30 minutes, followed by natural filtration (size 125 mm filter paper: No. 2) to remove solids. This operation was repeated three times to recover about 290 ml of methanol extraction filtrate. The filtrate was concentrated under reduced pressure, and the solvent was distilled off. Finally, in order to completely distill off the solvent, a vacuum freeze-drying treatment was performed to obtain about 360 mg (skin part) and 250 mg (edible part) of a dried taro extract. The skin extract was DMSO (dimethylsulfoxide), and the edible portion extract was adjusted to a concentration of 10 mg / ml with methanol to obtain an extracted sample.

(2) HCV replicon RNA production suppression test
(2-1) HCV replicon cytotoxicity test 90 μl each of suspension of test cells (replicon cells) (5 × 10 ⁴ cells / ml) was added to a transparent 96-well plate at 37 ° C. in the presence of 5% CO ₂ . The cells were cultured for 24 hours under conditions of 100% relative humidity. Seven-fold dilution preparation in a 3-fold dilution series so that the final concentration of the extracted sample prepared in (1) is 100 μg / ml in the edible part and 50 μg / ml in the skin, and 10 μl / well on the 96-well plate. Added. Thereafter, the cells were further cultured for 72 hours. Once the culture solution was discarded, 100 μl / well of Cell counting Kit-8 reagent (Dojindo Laboratories Co., Ltd.) diluted 10-fold with phosphate buffer (PBS (−)) was added, followed by further incubation for 4 hours. After culturing, the absorbance (450 nm) of the lysed culture solution at each concentration of the extracted sample was measured with an absorption microplate reader (trade name: EMax ^™ manufactured by Nihon Molecular Device Co., Ltd.) using 650 nm as a control wavelength. As a control, the absorbance of a lysed culture prepared using DMSO or methanol, which is a solvent used for preparing the extracted sample from the dried extract, was measured instead of the above-described extracted sample.
From the absorbance measurement value, the percentage with respect to the control was obtained, and the number of living cells (%) of the test cells at each concentration of the extracted sample was calculated. The value at which the number of viable cells reached 50% was defined as C ₅₀ (50% viable cell concentration: μg / ml). The result of the edible part methanol extraction sample is shown in FIG. 1, and the result of the peel methanol extraction sample is shown in FIG.

(2-2) HCV Replicon RNA Production Inhibition Test 90 μl each of suspension (5 × 10 ⁴ cells / ml) of test cells (replicon cells) was added to a white 96-well plate, and at 37 ° C. in the presence of 5% CO _2. The cells were cultured for 24 hours at 100% relative humidity. Six-fold dilution preparation was performed in a 4-fold dilution series so that the most concentrated final concentration of the extracted sample was 50 μg / ml, and added to the 96-well plate at a rate of 10 μl / well. Thereafter, the cells were further cultured for 72 hours. After removing the plate from the incubator and allowing it to stand at room temperature for 30 minutes or longer, a luciferase assay reagent (Promega, trade name:
Steady-Glo ^™ Luciferase Assay System) was added at 100 μl / well and well pipetted. After leaving it to stand for 5 minutes or more, light emission measurement was performed with a light emission detector (trade name: DTX800 manufactured by BECKMAN COULTER Co., Ltd.). As a control, the amount of luminescence was measured in the same manner for a reaction solution prepared in the same manner as described above using sterilized water instead of the extracted sample.

From the luminescence measurement value, the percentage relative to the control was obtained, and the luciferase activity (%) of the test cells at each concentration of the extracted sample was calculated. As described above, the luciferase activity (%) reflects the amount of HCV replicon RNA. The value at which the luciferase activity was 50% was defined as L ₅₀ (50% luciferase activity concentration: μg / ml).

The ratio of the C ₅₀ of the viable cell number to the L ₅₀ of the luciferase activity was defined as the selectivity index (SI = viable cell number C ₅₀ / luciferase activity L ₅₀ ). A larger value means that HCV replicon RNA production is suppressed without damaging the cells. The result of the edible part methanol extraction sample is shown in FIG. 1, and the result of the peel methanol extraction sample is shown in FIG.

<Comparative Example 1>
Preparation of IFN sample and inhibition test of HCV replicon RNA production Interferon (Funakoshi Co., Ltd., trade name: IFN-α2 (α2b), Human, Recombinant) was dissolved in PBS (with 0.1% BSA added). Ten-fold dilution preparation was carried out in a 2-fold dilution series with the highest final concentration of 10 U / ml, and an HCV replicon RNA production suppression test was conducted according to Experimental Example 1. Luciferase and WST-8 assays were performed 48 hours after adding the IFN reagent. The results are shown in FIG.

Taro Sennan Nakano methanol extract samples early edible portion is _{C 50} number of viable cells as shown in FIG. 1 is 100 [mu] g / ml or more, _{L 50} of the luciferase activity were 3.25μg / ml, SI is 30 or more. Methanol extract sample peels _{C 50} in the number of viable cells as shown in FIG. 2 41.59μg / ml, _{L 50} of luciferase activity 0.49μg / ml, SI is 85, one of the edible portion, skin In HCV, an inhibitory effect on HCV replicon RNA production was also observed. Further, at the concentration of edible part methanol extracted sample of 100 μg / ml, the relative viable cell number was almost the same as the control, and the relative luciferase activity was suppressed to 0.08%. In the peeled methanol extract sample, the concentration of 5.56 μg / ml, the number of relative viable cells was almost the same as the control, and the relative luciferase activity was suppressed to 0.92%. These effects are comparable to those of IFN (FIG. 3).

<Experimental example 2>
We examined the difference between the varieties using 8 varieties of Sennen Nakano, Sachi Fuji, Saku Daikichi, Akabane Daikichi, Aoi Ono, Saya Onna, Kyoen, and Eguimono.

(1) Preparation of taro methanol extraction sample The above-mentioned eight varieties were used as test samples. A methanol extraction sample was prepared according to Experimental Example 1. The extract was dissolved in methanol and used as an extraction sample.

(2) HCV replicon RNA production suppression test According to Experimental Example 1, an HCV replicon RNA production suppression test was performed. The results of the edible portion methanol extracted sample are shown in Table 1 and FIG. 4, and the results of the peel methanol extracted sample are shown in Table 2 and FIG.

Any of varieties _{C 50} by edible part is a 100 [mu] g / ml or _{more, L 50} was 3.25~63.37μg / ml.

C ₅₀ is a 25.26~133.01μg / _{ml, L 50} was 1.72~3.62μg / ml. In the SI value, white bud Daikichi was the highest value.

  As can be seen from Table 1, Table 2, FIG. 4 and FIG. 5, the tuber extract of the taro family plant has an HCV replicon RNA production inhibitory action in any cultivar, and the skin extract has a stronger HCV. It was revealed that a replicon RNA production inhibitory action was observed.

<Experimental example 3>
(1) Preparation of 80% ethanol extraction sample of taro Sennan Nakano precocious Dry powder of taro Sennan Nakano precocious skin was obtained according to Experimental Example 1. To about 1 g of the obtained dry powder of taro skin, about 30 ml of 80% by volume ethanol aqueous solution was added and stirred vigorously at room temperature for 30 seconds, followed by suction filtration (Kiriyama Mfg. Co., Ltd., trade name: Kiriyama funnel Size 60 mm 5A) was performed to remove solids. The filtrate was concentrated under reduced pressure, and the solvent was distilled off. Finally, in order to completely distill off the solvent, vacuum lyophilization treatment was performed to obtain about 100 mg of a dried extract of taro skin. This was adjusted to a concentration of 200 mg / ml with DMSO and used as an extraction sample.

(2) HCV Replicon RNA Production Inhibition Test Nine-stage dilution preparation was performed in a 3-fold dilution series with the highest final concentration of 500 μg / ml, and an HCV replicon RNA production inhibition test was conducted according to Experimental Example 1. The result of the skin 80% ethanol extraction sample prepared in (1) is shown in FIG.

C ₅₀ was 99 μg / ml and L ₅₀ was 5 μg / ml. The SI value was 20, and the HCV replicon RNA production inhibitory activity was recognized even with 80% ethanol extract of skin.

<Experimental example 4>
(1) Northern blot analysis Test cells (replicon cells) were seeded on a 12-well plate to 4 × 10 ⁴ cells / well, and cultured for 24 hours at 37 ° C. in the presence of 5% CO ₂ and a relative humidity of 100%. . The skin extract of Sennan Nakano Wassei was diluted in four stages in a 3-fold dilution series so that the darkest final concentration was 50 μg / ml, and this diluted series was dispensed on a test plate by 100 μL / well. Thereafter, the cells were further cultured for 72 hours. The culture solution was discarded and stored at -80 ° C.

  A 12-well plate stored at −80 ° C. was taken out, and RNA was collected by Rneasy mini kit (Qiagen). The obtained RNA was quantified by Ultraspec 3300. A sample was prepared to 2 μg / lane, 3 × Sample Buffer was added, treated at 65 ° C. for 15 minutes, and then rapidly cooled. Electrophoresis (100V, 60 minutes) with 1% agarose gel, the obtained gel was stained with ethidium bromide solution, and after confirming the band, the membrane was used with Northern Max (Ambion brand name: Formaldehyde Load Dye Northern Max) (Ambion brand name: Northern Max Transfer Buffer). The membrane was washed with TBE, prehybridized at 42 ° C., and hybridized with a neomycin resistant gene biotinylated probe contained in the replicon at 42 ° C. overnight (Ambion product name: ULTRAhyb). The membrane was washed with a wash buffer, blocked (Aloka product name: Blocking Buffer), and detected with streptavidin-Alexa Fluor 680 conjugate (Molecular probes) with Odyssey (Aloka).

The IC50 value was calculated based on the density of the detected band. The detection results are shown in Table 3 and FIG.

  In Northern blotting, the IC50 value was 4.4 μg / ml, and HCV RNA almost disappeared at 16.6 μg / ml or more.

<Experimental example 5>
(1) Western blot analysis According to Experimental Example 4, cells were prepared using a 12-well plate. A 12-well plate stored at −80 ° C. was taken out, and the cells were removed by pipetting with 1 mL of PBS and collected in a tube. After centrifugation at 10,000 rpm for 3 minutes, the supernatant was aspirated, and 1% Protease Inhibitor (SIGMA brand name: PROTEASE INHIBITOR COCKTAIL) Lysis Buffer (SIGMA brand name: CelLyticTM-M) was added to lyse the cells. . The mixture was shaken for 15 minutes, centrifuged at 15,000 rpm for 15 minutes, the supernatant was collected, and protein quantification was performed by the Bradford method.

  After protein quantification, a sample was prepared to 5 μg / lane, and 4 × Sample Buffer (WAKO, Inc., trade name: Sample Buffer Solution (2ME +) (× 4)) was added to one-fourth volume and 95 ° C. for 5 minutes. Heat treatment was performed. SDS-PAGE was performed at 30 mA for 60 minutes, and the obtained gel was blotted at 50 V overnight. After the membrane was washed with TBS, blocking was performed for 30 minutes, and a primary antibody (Roche α-NS3) was added and reacted for 30 minutes. After washing twice with TBST, the reaction was carried out for 30 minutes with a secondary antibody (Molecular probes, trade name: Alexa 680 conjugated anti-rabbit IgG (Goat)). After washing, detection was performed by Odyssey. The detection results are shown in Table 4 and FIG.

  In Western blotting, the IC50 value was 1.9 μg / ml, and HCV protein (NS3) almost disappeared at 50 μg / ml or more.

  An HCV production inhibitor comprising as an active ingredient a processed product of a tuber of a taro plant of the present invention, for example, a pulverized product of a taro plant tuber, a juice of a tuber, and an extract of a tuber, is intended for an HCV-infected person, To prevent and / or treat HCV-induced diseases, such as the development of hepatitis, acute and chronic hepatitis in HCV infected people, and diseases that develop from chronic hepatitis to cirrhosis and liver cancer, or worsened by viral infections Is preferred. In addition, since the tuber of a naturally derived taro plant that can be originally used for food is used as an active ingredient, it has few side effects and is highly usable as a daily medicine that can be safely taken for a long time.

It is a graph which shows the result of the HCV replicon RNA production suppression test and cytotoxicity test by the taro sennan-nakano early edible part methanol extraction sample. It is a graph which shows the result of the HCV replicon RNA production suppression test and the cytotoxicity test by the taro Sennan Nakano early life skin methanol extraction sample. It is a graph which shows the result of the HCV replicon RNA production suppression test and cytotoxicity test by interferon. It is a graph which shows the result of the HCV replicon RNA production suppression test by eight kinds of taro edible part methanol extraction samples. It is a graph which shows the result of the HCV replicon RNA production suppression test by eight kinds of taro skin methanol extraction samples. It is a graph which shows the result of the HCV replicon RNA production suppression test and cytotoxicity test by the 80% ethanol extraction sample of taro sennan-nakano. It is the figure which measured HCV replicon RNA by the northern blot analysis using the taro Sennan-nakano early life skin methanol extraction sample. It is the figure which measured HCV replicon RNA by the Western blot analysis using the taro Sennan Nakano early life peel methanol extraction sample.

Claims (12)

  A hepatitis C virus production inhibitor comprising a processed tuber of a taro plant as an active ingredient.   The hepatitis C virus production inhibitor according to claim 1, wherein the processed product of the taro family plant is at least one selected from the group consisting of a crushed tuber, a squeezed tuber, and a tuber extract.   The hepatitis C virus production inhibitor according to claim 1 or 2, wherein the processed product of the taro family plant is tuber skin.   The pharmaceutical composition containing the hepatitis C virus production inhibitor according to claim 1, which is used for suppressing virus production for a person infected with hepatitis C virus.   The hepatitis C according to claim 1, which is used for preventing and / or treating a disease induced by hepatitis C virus or a disease aggravated by these virus infections for a person infected with hepatitis C virus. A pharmaceutical composition comprising a virus production inhibitor.   The pharmaceutical composition containing the hepatitis C virus production inhibitor according to claim 1, which is used for suppressing the onset of hepatitis induced by hepatitis C virus for a person infected with hepatitis C virus.   A hepatitis C virus production suppression material comprising a processed tuber of a taro plant as an active ingredient.   The hepatitis C virus production suppression material according to claim 8, wherein the processed product of the taro family plant is at least one selected from the group consisting of a pulverized tuber, a tuber juice, and a tuber extract.   The hepatitis C virus production suppression material according to claim 8 or 9, wherein the processed product of the taro family plant is a tuber skin.   The food composition containing the hepatitis C virus production suppression material of Claim 7 used in order to suppress the production of a virus with respect to a hepatitis C virus infected person.   The hepatitis C virus according to claim 7, which is used for preventing and / or treating a disease induced by hepatitis C virus or a disease worsened by these virus infections for a person infected with hepatitis C virus. A food composition comprising a virus production-suppressing material.   The food composition containing the hepatitis C virus production suppression material of Claim 7 used in order to suppress the onset of the hepatitis induced by the hepatitis C virus with respect to hepatitis C virus infection.