KR101708833B1 - Anti-viral composition comprising curcumin - Google Patents

Abstract

The present invention relates to an antiviral composition comprising curcumin which can be used for the treatment and prevention of non-bacterial food poisoning and can contribute to public health improvement by reducing non-bacterial food poisoning

Description

Anti-viral composition comprising curcumin < RTI ID = 0.0 >

The present invention relates to an antiviral composition, and more particularly to an antiviral composition comprising curcumin, a pharmaceutical composition for preventing or treating non-bacterial food poisoning comprising curcumin as an active ingredient, And a health functional food for preventing or improving non-bacterial food poisoning.

Norovirus is known worldwide as a causative agent of non-bacterial acute gastroenteritis and is a type of enteric virus that causes infectious gastroenteritis in humans when food or water is ingested. These Noroviruses belong to the family Caliciviridae and have a signle-stranded RNA genome of 7.5 kb in size. It consists of three open reading frames (ORFs).

Norovirus was first detected in 1968 in Norwalk Elementary School in Ohio, USA, in the case of diarrhea in a group of food poisoning patients, and at that time (Norwalk Viruses). Subsequently, viruses similar to the Norwalk virus were found in similar food poisoning patients, and were named after the region they were found, such as the Hawaiian virus, the Montgomery virus, and the Townton virus. It was also known as a small round-structured virus (SRSV) in that the virus was small and globular among the viruses in 1972 under an electron microscope. The virus was then isolated from patients with non-bacterial acute gastroenteritis, Because the virus was found one after another, it was also temporarily called Norwalk-like virus. In August 2002, the International Committee on Taxonomy of Viruses (ICTV) unified the name Norovirus. Three of the five norovirus types (GI, G II, G IV) of five types (GI, GII, GIII, GIV, GV) classified by genotype have been found to be infectious to humans (CDC , 2010).

This is a major cause of food poisoning that can occur in all age groups. It is known to cause more than 90% of non-bacterial enteritis. Norovirus is a virus that can survive in the cold and dry winter months. It has a physically and chemically stable structure. It can survive for 10 days at room temperature, 10 ℃ sea water for 30-40 days, have. It can be transmitted to humans through food and water, or through direct contact with the patient or air. Even ingesting 10 particles of Norovirus can cause food poisoning, and it is infectious enough to spread for 2 weeks even after symptoms disappear.

Infections with Norovirus cause nausea, vomiting, diarrhea and abdominal pain. In most cases, the symptoms are mild and recover naturally after 1 to 2 days. The incubation period is from 24 to 48 hours. Viruses in the stool or vomit of the infected person are contaminated with food or water, or contaminated with food, food, etc., contacted by the infected person. It spreads easily enough to be easily infected even with a small amount of virus, and the infectious disease is most severe during the period of symptoms and lasts from 3 days to up to 2 weeks after recovery (Health Information of Disease Control Division).

In the United States, 48 million people, or one in six Americans, die from food poisoning each year, with 128,000 hospitalizations and 3,000 deaths. Ninety percent of food poisoning cases leading to death are due to norovirus pathogens (CDC December 15, 2010). In Korea, the number of patients suffering from norovirus food continues to increase, accounting for about 30% of all food poisoning cases except for 2009, which was influenced by the H1N1 influenza virus since 2006. In addition to pathogenic Escherichia coli, It is counted as a cause.

In particular, human norovirus is proliferating only in the intestines of humans, and since cell culture is impossible to date, related studies are insufficient and vaccine and therapeutic agent have not been developed yet. However, as a surrogate for murine norovirus has become possible to culture in RAW 264.7 cells, related virus studies have become active. RAW 264.7 cells are mouse leukemic monocyte macrophage cell lines from STAT1 deficient (STAT1 - / -) immunocompromised rats and are the only cells in which murine norovirus-1 (MNV-1) System.

Norovirus has a variety of genotypes, but research on several cell strains is still limited because it is difficult to cultivate virus cells. Therefore, it is difficult to develop a vaccine and a drug, and research on various strains capable of culturing Norovirus has been limited, and various researches on a substance inhibiting Norovirus have not been conducted.

In addition, the conventional method of inhibiting norovirus is mainly based on physicochemical methods. Interest in developing norovirus inhibitors using natural substances harmless to the human body due to the basic investment cost and the negative effect on synthetic chemicals Is increasing.

Accordingly, the present inventors have made intensive researches to overcome the problems of the prior arts, and as a result of screening for norovirus by screening inhibitors that inhibit cell-based virus activity, curcumin has a high antiviral activity Respectively.

Thus, an example of the present invention provides an antiviral composition against norovirus comprising curcumin.

Another example is to provide a pharmaceutical composition for prevention and / or treatment of non-bacterial food poisoning comprising curcumin.

Another example is to provide a health functional food for prevention and / or improvement of non-bacterial food poisoning including curcumin.

In order to achieve the above-mentioned object, the present invention provides a novel norovirus (norovirus) containing curcumin which is excellent in inhibitory effect against norovirus, which is a major virus mediating non-bacterium food poisoning, A pharmaceutical composition for preventing or treating non-bacterial food poisoning, and a health functional food for prevention or improvement of non-bacterial food poisoning.

Hereinafter, the present invention will be described in more detail.

One example of the present invention relates to an antiviral composition for curcumin against norovirus. In the above antiviral composition, curcumin is included as an effective ingredient for exerting an antiviral effect on norovirus, and can be administered in a pharmaceutical effective amount capable of exerting an antiviral effect on norovirus, for example, a virus growth and / ≪ / RTI >

Curcumin has the formula C ₂₁ H ₂₀ O ₆ and has a molecular weight of 368.38. It is a yellow spice of polyphenol which has been widely used in Indian food. It is turmeric curcuminoid. It is used as yellow pigment in food additive and is used as spice. Curcumin contains two tautomers, keto and enol, by the keto-enol tautomerism. In addition, curcumin inhibits the early oncogenic process of azoxymethane (AOM) induced colorectal cancer, inhibits the formation of DNA adduct by benzopyrene, and has anticarcinogenic and antimutagenic activities.

The curcumin may be synthesized, isolated, isolated or fractionalized, but is not limited thereto.

The antiviral composition according to the present invention may exhibit antiviral activity by inhibiting the replication and replication of norovirus. The antiviral composition may be in the form of various detergents or disinfectants that can be administered to mammals including vertebrate animals, preferably humans, such as hand cleansing agents or the like, or may be in the form of disinfectants, food additives that can be added to foods, The present invention can be applied to all kinds of conventional antiviral agents such as conventional cleaning agents and disinfectants which can be applied to various kinds of articles.

The content of curcumin as an active ingredient contained in the antiviral composition may be appropriately determined depending on the form of the composition, the desired use, and the like, for example, 0.1 to 10 mg / ml, 0.1 to 5 mg / ml, ml, 0.25 to 5 mg / ml, 0.25 to 3 mg / ml, 0.5 to 10 mg / ml, 0.5 to 5 mg / ml, 0.5 to 3 mg / ml, 0.75 to 10 mg / ml , 0.75 to 5 mg / ml, 0.75 to 3 mg / ml, 1 to 10 mg / ml, 1 to 5 mg / ml, or 1 to 3 mg / ml.

Another example of the present invention relates to a pharmaceutical composition for the prophylaxis and / or treatment of non-bacterial food poisoning comprising curcumin or the antiviral composition. The non-bacterial food poisoning is a viral food poisoning caused by infection of the virus, and more specifically, it may be all diseases and / or symptoms related to food poisoning caused by norovirus infection, for example, ), Diarrhea, or fever.

In the pharmaceutical composition, curcumin is included as an effective ingredient for exerting preventive and / or therapeutic effects of non-bacterial food poisoning, and may be contained in a pharmaceutically effective amount capable of exhibiting a desired prevention and / or therapeutic effect of non-bacterial food poisoning.

The pharmaceutical composition according to the present invention can be administered to mammals including human in various routes. The mode of administration may be any conventionally used route and may be administered, for example, orally, or by parenteral routes such as skin, intravenous, intramuscular, subcutaneous, and the like, preferably orally.

The pharmaceutical compositions according to the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols, or in the form of transdermal preparations, suppositories, Parenteral formulations, and the like.

The pharmaceutical composition of the present invention may be pharmaceutically acceptable and may further contain adjuvants such as physiologically acceptable carriers, excipients and diluents. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used.

When the composition for parenteral administration of the pharmaceutical composition according to the present invention is provided, it is possible to provide a composition for parenteral administration such as a liquid, a gel, a cleansing composition, a tablet for insertion, a suppository form, cream, ointment, dressing solution, spray, And may be a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, a suppository, a cream, an ointment, a jelly, a foam, a cleanser or an insert A skin external preparation such as a liquid preparation, a gel preparation, a cleaning composition, and a tablet for insertion may be included. The formulation can be prepared, for example, by adding a solubilizer, an emulsifying agent, a buffering agent for pH control, etc. to the sterilized water. Examples of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

More particularly, the pharmaceutical composition may comprise a carrier for formulating it in addition to the antiviral composition or curcumin. The carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersant, a suspending agent,

In embodiments where the pharmaceutical composition of the present invention is applied to humans, the pharmaceutical composition of the present invention may be administered alone, but is generally administered in a pharmacological manner selected in consideration of the mode of administration and standard pharmaceutical practice May be administered in admixture with a carrier. For example, the pharmaceutical composition of the present invention may be in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or as an excipient, or as an elixir or a suspending agent containing a flavoring or coloring agent 0.0 > oral, < / RTI > intraoral, or sublingually.

The content of curcumin as an active ingredient contained in the pharmaceutical composition is not particularly limited depending on the form of the composition, the intended use, etc., and is in the range of 0.1 to 10 mg / ml, 0.1 to 5 mg / ml, 0.1 to 3 mg / ml , 0.25 to 10 mg / ml, 0.25 to 5 mg / ml, 0.25 to 3 mg / ml, 0.5 to 10 mg / ml, 0.5 to 5 mg / ml, 0.5 to 3 mg / 0.75 to 5 mg / ml, 0.75 to 3 mg / ml, 1 to 10 mg / ml, 1 to 5 mg / ml, or 1 to 3 mg / ml.

The dose of the pharmaceutical composition of the present invention may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient, and may be determined by a doctor or pharmacist once, It may be administered in divided doses. For example, a daily dose of 0.001 to 10000 mg / kg, 0.01 to 10000 mg / kg, 0.1 to 10000 mg / kg, 0.5 to 10000 mg / kg, 0.001 to 1000 mg / kg, kg, from 0.01 to 1000 mg / kg, from 0.1 to 1000 mg / kg, from 0.5 to 1000 mg / kg, from 0.001 to 500 mg / kg, from 0.01 to 500 mg / kg, , 0.001 to 300 mg / kg, 0.01 to 300 mg / kg, 0.1 to 300 mg / kg, or 0.5 to 300 mg / kg. The above-mentioned dosage is an average case, and the dose may be high or low depending on individual differences.

If the daily dosage of the pharmaceutical composition of the present invention is less than the above dose, a significant effect can not be obtained, and if it exceeds the above range, it is not only economical but also causes an undesirable side effect It is preferable to set the above range.

Another example of the present invention relates to a health functional food for preventing and / or improving non-bacterial food poisoning which contains curcumin as an active ingredient. The health functional food may be used to include various foods, food additives, and health drinks.

The content of curcumin as an active ingredient contained in the health functional food is not particularly limited depending on the form of the food and the intended use, and is, for example, 0.00001 to 100% by weight, 0.01 to 100% by weight, 0.01 to 50% by weight, 0.01 to 50% by weight, 0.00001 to 15% by weight and 0.01 to 15% by weight, But are not limited to, 1 to 50% by weight, 0.01 to 15% by weight, 1 to 15% by weight, 0.01 to 10% by weight or 1 to 10% by weight.

In the health functional food of the present invention, there is no particular limitation on the liquid component other than the curcumin as an essential ingredient in the indicated ratio in the beverage, and various kinds of flavors or natural carbohydrates, .

Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavoring agents (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0.01 to about 0.2 parts by weight per 100 parts by weight of the health functional food of the present invention.

Another example of the present invention relates to a cleaning product for preventing and / or improving non-bacterial food poisoning which contains curcumin as an active ingredient. The cleaning product may be a solid cosmetic soap, a hand cleaner, a liquid shampoo, a liquid soap, a liquid rinse, a body cleaner, a creamy soap, or the like.

The present invention relates to antiviral compositions against norovirus, and more particularly to antiviral compositions against norovirus comprising curcumin. The present invention may be used for non-bacterial food poisoning treatment and / or prevention and is expected to contribute to public health improvement by reducing non-bacterial food poisoning.

1 is a graph showing the cytotoxicity test results of a composition containing curcumin as an active ingredient on Raw 264.7 cells according to an embodiment of the present invention.
2 is a graph showing the antinorovirus activity according to the concentration of a composition containing curcumin as an active ingredient according to an embodiment of the present invention.
Figure 3 is a graph showing the results for short-term (10, 30, 60, and 120 min) anti-norovirus activity of a composition comprising curcumin as an active ingredient according to one embodiment of the present invention.
FIG. 4 is a graph showing the results for long-term (1, 3, 7, and 14 days) anti-norovirus activity of a composition containing curcumin as an active ingredient according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One. Curcumin  Preparation of composition samples

A sample of the composition containing curcumin as an active ingredient was added to 1 ml of a cell culture-exclusive dimethyl sulfoxide (DMSO) as a solvent, 0.5 ml of curcumin (available from Sigma Aldrich (USA) HPLC) were respectively dissolved in 0.25 mg, 0.5 mg, 0.75 mg, 1 mg and 2 mg to prepare respective composition samples containing curcumin as an active ingredient at concentrations of 0.25, 0.5, 0.75, 1 and 2 mg / mL.

Example  2. Cytotoxicity measurement

The cytotoxicity test of the composition sample containing curcumin prepared in Example 1 was carried out by measuring the cell viability using an MTT assay (Applied Microbiology, 2009, 49, 806-808).

Specifically, RAW 264.7 cells (KCLB No.40071, Korean Cell Line Bank) were seeded at 1.5 × 10 ⁶ cells / ml per well in 96 wells, and 12 hours later, samples of Example 1 were added in concentration- 1% NEAA (nonessential amino acid), 1.5% sodium bicarbonate, 1% HEPES (2- [4- (2-hydroxyethyl) piperazin- were cultured in Dulbecco's modified Eagle's Medium (Gibco, Grand Island, NY) containing 1% (v / v) PS (Gentamycin 1% (v / v)) for 1 hour at 37 ° C in a 5% CO ₂ incubator.

Then, MTT was added to a final concentration of 0.5 mg / ml. After 2 hours, absorbance (MTT assay) was measured at 570 nm on a UV-Vis spectrophotometer (SpectraMax M2, Molecular Devices Corp. USA). The cell viability was calculated as a percentage relative to the no-added group in which the composition sample was not added. The results are shown in Table 1 and Fig.

Curcumin concentration (mg / mL) Cell survival rate (%) 0.25 96.31 0.5 103.12 0.75 99.83 1.0 97.02 2.0 97.44

As can be seen from the above Table 1, it was confirmed that there was no cytotoxicity at the concentrations of curcumin 0.25, 0.5, 0.75, 1 and 2 mg / mL.

Comparative Example . Curcumin  Untreated control group

Raw 264.7 cells were inoculated with DMSO at a temperature of 4 ° C for 3 days (Herbert W. Virgin at the Washington University School of Medicine) (virus concentration: 1.08 × 10 ⁵ ), 1.5% (w / v) sea plaque agarose and 2X-Minimum Essential Medium (MEM) at a ratio of 1: 1, and cultured in a 37 ° C incubator for 72 hours to prepare a control.

Example  3. Curcumin  Depending on concentration Antinorovirus  Active measurement

Plaque assay was performed to investigate the antinorovirus activity according to curcumin concentration.

Concretely, the curcumin-containing sample of Example 1 was mixed with Murine Norovirus (available from Herbert W. Virgin at the Washington University School of Medicine), and the mixture was reacted at 4 ° C for 72 hours. Then, the mixture : 1.08 χ 10 ⁵ ) were inoculated into Raw 264.7 cells and mixed with 1.5% (w / v) sea plaque agarose and 2X-MEM medium at a ratio of 1: 1 and cultured in a 37 ° C. incubator for 72 hours The degree of virus inhibition was confirmed, and the antinorovirus activity was measured according to the concentration of curcumin.

The degree of virus inhibition was measured by counting the plaque in pfu / mL of the digestion broth by staining with 0.5% (v / v) crystal violet for each concentration, and the number of plaques in the untreated group was measured in pfu / mL Plaque was counted, and the value of the curcumin-treated group versus the curcumin-treated group was represented by log reduction. The log reduction value indicates how much the curcumin composition was inhibited by each concentration compared with the control group. The larger the log reduction value, the greater the effect of the curcumin composition at the concentration. The results are shown in Table 2 and FIG.

Concentration of curcumin
(mg / mL) Measured value of virus inhibition degree
(Log) 0.25 -0.3228 0.5 -0.4996 0.75 -0.5081 1.0 -0.9996 2.0 -1.0191

As can be seen in Table 2 and FIG. 2, it was confirmed that the number of plaques was significantly reduced at the concentrations of 0.25, 0.5, 0.75, 1 and 2 mg / mL. Especially, at concentrations exceeding 0.75 mg / It was confirmed that norovirus activity was present.

Example  4. Short-term Antinorovirus  Active measurement

A plaque assay was performed to determine the short term antinorovirus activity of curcumin.

Specifically, Murine Norovirus (available from Herbert W. Virgin at the Washington University School of Medicine) was mixed with a sample having a curcumin concentration of 1 mg / mL in the sample of Example 1, and the mixture was treated for 10 minutes, 30 minutes, 60 (V / v 1.08 × 10 ⁵ ) were inoculated into Raw 264.7 cells and 1.5% (w / v) sea plaque agarose and 2X-Minimum Essential Medium medium were added to 1 : 1, and incubated for 72 hours at 37 ° C in an incubator to determine the degree of virus inhibition, and the curcumin-mediated short-term antiviral activity was measured.

The degree of virus inhibition was measured by 0.5% (v / v) crystal violet at each hour, and the plaque was counted in pfu / mL of the digestion medium, and plaques were collected in units of pfu / mL The values of the curcumin treated group versus the non-treated group were expressed by log reduction. The results are shown in Table 3 and FIG.

Culture time (min) Measured value of virus inhibition degree (Log) 10 -0.1775 30 -0.1955 60 -0.3834 120 -0.6995

As can be seen in FIG. 3, the results for curcumin's short-term antinorovirus activity showed a significant decrease in the number of plaques over time, with results corresponding to 10, 30, 60, and 120 minutes. It can be seen that the activity of curcumin is increased by the passage of time.

Example  5. Long term Antinorovirus  Active measurement

A plaque assay was performed to determine the long-term antinorovirus activity of curcumin.

Specifically, a sample having a curcumin concentration of 1 mg / mL in the sample of Example 1 was mixed with Murine Norovirus (available from Herbert W. Virgin at the Washington University School of Medicine) days and after 14 days reaction, the mixture (virus concentration: 1.08 ⅹ 10 ⁵⁾ a and inoculated in Raw 264.7 cells, 1.5% (w / v) agarose (sea plaque agarose) and 2X-MEM a medium 1: 1 , And incubated for 72 hours at 37 ° C in an incubator to determine the degree of virus inhibition and to measure the long-term antiviral activity of curcumin.

The degree of virus inhibition was measured by 0.5% (v / v) crystal violet at each hour, and the plaque was counted in pfu / mL of the digestion medium, and plaques were collected in units of pfu / mL The values of the curcumin treated group versus the non-treated group were expressed by log reduction. The results are shown in Table 4 and FIG.

Culture time (days) Measured value of virus inhibition degree One -0.6783 3 -1.0287 7 -0.7404 14 -0.6613

As can be seen in FIG. 4, the results of long-term antinorovirus activity of curcumin did not decrease in a time-dependent manner for days 1, 3, 7, and 14, but in the case of day 3, It was confirmed that the virus was reduced to 1 log ₁₀ or more, that is, the number of living viruses was reduced to 1/10 or less. It can be seen that the activity of curcumin is increased by the passage of time.

As can be seen from Examples 3 to 5, it can be seen that the antiviral activity of curcumin, which can be easily ingested by foods such as beverages, is particularly excellent. Thus, a composition comprising curcumin according to one embodiment of the present invention is a virus Functional food, and cleansing products, which are effective for prevention and / or treatment of food poisoning.

Claims (15)

delete delete delete A pharmaceutical composition for the prevention or treatment of symptoms of non-bacteriological food poisoning caused by infection with norovirus comprising curcumin as an active ingredient, wherein the symptoms of the bacteriophilic food poisoning include noroviral gastroenteritis, vomiting, nausea, And pyrogen. ≪ / RTI > delete delete 5. The pharmaceutical composition according to claim 4, wherein the curcumin is contained at a concentration of 0.1 to 10 mg / mL. A health-functional food for preventing or ameliorating the symptoms of non-bacteriological food poisoning caused by infection with norovirus comprising curcumin as an active ingredient, wherein the symptoms of the bacteriophilic food poisoning include noroviral gastroenteritis, vomiting, nausea, And fever. ≪ / RTI > delete delete 9. The health functional food according to claim 8, wherein the curcumin is contained in an amount of 0.01 to 15% by weight based on the solid content. The health functional food according to claim 8, wherein the health functional food is a beverage, a fermented milk, or a food additive. A cleansing product for preventing or ameliorating the symptoms of non-bacteriological food poisoning caused by infection with norovirus comprising curcumin as an active ingredient, wherein the symptoms of the bacteriophilic food poisoning include noroviral gastroenteritis, vomiting, nausea, Characterized in that it is characterized by at least one selected from the group consisting of: 14. The article of claim 13, wherein the cleaning article is a solid cosmetic soap, a hand cleaner, a liquid shampoo, a liquid soap, a liquid rinse, a body cleanser, or a creamy soap. delete

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