CN107073057B - Composition for preventing and treating viral diseases containing Epimedium koreanum extract as active ingredient - Google Patents

Composition for preventing and treating viral diseases containing Epimedium koreanum extract as active ingredient Download PDF

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CN107073057B
CN107073057B CN201580055424.0A CN201580055424A CN107073057B CN 107073057 B CN107073057 B CN 107073057B CN 201580055424 A CN201580055424 A CN 201580055424A CN 107073057 B CN107073057 B CN 107073057B
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virus
extract
cells
epimedium koreanum
vesicular stomatitis
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CN107073057A (en
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马珍烈
赵元卿
金兑洙
李宗洙
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Korea Institute of Oriental Medicine KIOM
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • A61K36/296—Epimedium
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating viral diseases, which comprises an extract of Epimedium koreanum (Epimedium koreanum) as an active ingredient. It was found that the epimedium koreanum extract of the present invention increases the cell survival rate of cells infected with viruses, such as vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus, and reduces the number of viruses in the cells. The epimedium koreanum extract of the present invention inhibits virus proliferation in cells, and thus can be effectively used as a pharmaceutical composition for preventing and treating viral diseases with respect to vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus.

Description

Composition for preventing and treating viral diseases containing Epimedium koreanum extract as active ingredient
Technical Field
The present invention provides a pharmaceutical composition for preventing and treating viral diseases, which comprises an extract of Epimedium koreanum nakai as an active ingredient.
Background
Vesicular Stomatitis Virus (VSV) is the viral pathogen that causes vesicular stomatitis. The virus is largely divided into two serotypes (i.e., indiana and new jersey types). The new jersey type is known for its greater pathogenicity and has more pronounced lesions than the indiana type. As vesicular stomatitis occurs mainly in the continental americas, it is also referred to as a new continental disease. In the united states of america, outbreaks have occurred intermittently since 2004 and then each year. Southern Mexico, Central America countries, northern south America, Brazil and the like are representative regions where diseases frequently occur.
Vesicular stomatitis cannot be distinguished from foot-and-mouth disease, vesicular herpes, swine vesicular disease, and the like, and has only clinical symptoms showing vesicles, so rapid diagnosis in a laboratory is necessary. In the case of a disease with a significant clinical symptom, the disease is diagnosed by virus isolation from a pathogen or reverse transcription-polymerase chain reaction, etc., and in the absence of such clinical symptom, isolation of the pathogen is difficult, and thus serological diagnosis is performed. In the blood of host animals infected with vesicular stomatitis virus, specific antibodies are typically formed 5-8 days after infection. Methods for detecting antibodies include complement fixation assays, neutralization assays, enzyme linked immunoassays, and the like. Vesicular stomatitis affects many farm animals as a serious infectious disease classified by the world animal health Organization (OIE) as class a with foot-and-mouth disease, which causes enormous economic losses to affected countries. Farm animals that may be affected by vesicular stomatitis include almost every mammal, such as horses, cattle, sheep, goats, and pigs, and it has been confirmed that cold-blooded animals may also be infected. Mortality due to vesicular stomatitis is very low. However, in outbreaks in ruminants or pigs animals are rapidly depreciated by the formation of vesicles on the tongue, lips, oral mucosa, papillae and hoof surfaces. In addition, vesicular stomatitis virus can also be transmitted to humans, and it has been reported to cause diseases with mild influenza-like symptoms in humans.
Newcastle Disease Virus (NDV) causes Newcastle Disease (ND) and acute and virulent respiratory diseases, which are known to be one of the most important animal diseases worldwide, and it is designated as a first-class epidemic by korean authorities because its infection of non-immunized poultry results in a mortality rate of 100%. The disease is divided into two types, named asian and american. Acute asian types are caused by viruses with strong pathogenicity, while american types are caused by viruses with weak pathogenicity. Korea is an area where newcastle disease virus is present at any time, and there is a potential risk factor in view of active trade between korea and south asia, east asia, mainland china, and taiwan areas where newcastle disease virus is prevalent.
Infection of poultry is transmitted by air or oral routes, etc., such as secretions, excretions, respiratory excretions, direct contact with infected birds, etc. The main symptoms of the disease are cough, wheezing, loss of appetite, pale green diarrhea following pale yellow diarrhea, paralysis, spasms, twitching, torticollis, twisted wings and legs, and congestion in the trachea. Newcastle disease in poultry occurs in all age groups and is highly contagious. Infection of unvaccinated chickens resulted in a mortality rate of 100%, and the disease has been designated as a primary epidemic by korean authorities. The bureau publishes preventive notices every year, and outbreaks not only increase, but also spread across the country, causing significant damage to poultry farms. Today, there is no known treatment or therapy for newcastle disease.
Most human herpes viruses (HSV) have an infection rate of 70% to 80% or more in korea. Viruses are representative chronic viruses that cause asymptomatic infection once infected and remain infectious in their host for repeated inactivation and reactivation. Herpes viruses are also representative viruses that cause opportunistic infections, since viruses cause various diseases through reactivation caused by a dysfunctional immune system or an innate infection, and the prognosis of an infection may be fatal depending on the immune status of a patient. Currently, there is no vaccine for prophylaxis or treatment of seven human herpesviruses other than the live varicella-zoster virus (VZV) vaccine. In view of the increased organ and hematopoietic stem cell transplantation, there is interest in controlling reactivated herpes virus infections resulting from dysfunctional immune systems, such as human immunodeficiency virus (HIV-1) infections.
Existing anti-herpes virus agents have been developed primarily against the viral DNA polymerase (DNA pol). Among them, acyclovir and its derivatives are drugs that induce premature termination of DNA by insertion into the viral genome replicated by DNA pol after thymidine kinase (hereinafter referred to as "TK") specific phosphorylation of herpes virus, and are most commonly used in the past 40 years since development. In addition, drugs such as forskote (e.g., pyrophosphate analogs) are commonly used. These drugs are effective in treating alpha-herpes virus, but show limited clinical efficacy against beta-or gamma-herpes virus infection or the initial symptoms of reactivation infection (e.g. infectious mononucleosis). In particular, since there are no existing antiviral agents available for controlling gamma-herpes viruses, the treatment of gamma-herpes virus infections relies mainly on an allopathic therapy using steroid-type drugs.
Rhinoviruses belong to the genus Picornaviridae (Picornaviridae) and include sinusitis, asthmatic conditions, otitis media, cystic fibrosis, bronchitis, and the like. Human Rhinovirus (HRV) is known to be the major cause of acute exacerbations of asthma, and many stable asthmatic patients have human rhinovirus in their bronchial tissues. Comparing bronchial mucosa biopsy specimens from asthmatic patients and non-asthmatic patients, the frequency of human rhinovirus in the lower respiratory tract was significantly higher in the asthmatic group than in the non-asthmatic group, and a correlation between the presence of human rhinovirus and the clinical severity of asthma was also reported. Human rhinovirus-positive patients are known to show lower lung function, eosinophilia in bronchial mucosa, lymphocytosis, eosinophilic infiltration, and the like. Stable asthmatic patients without clinical symptoms of upper respiratory tract infections also showed an association of human rhinovirus with chronic inflammation in the lower respiratory tract.
Respiratory Syncytial Virus (RSV) is the major pathogen in lower respiratory tract infections, pneumonia and bronchitis (Brandt et al, Pediatrics 52:56 (1973); Selwyn et al, Rev infection Dis,12: s870 (1990)). In particular, the virus is high risk because the infection is lethal to infants with lung dysplasia, congenital heart disease, cystic fibrosis, cancer or various immunodeficiency, as well as adults with reduced immune function prior to bone marrow transplantation (Chandwani et al, J Pediatr,117:251 (1990); Bruhn et al, J Pediatr, 90:382 (1977)). The infection rate of elderly people is similar to the rate of influenza viruses, and it is reported that the excess mortality rate with RSV is higher than with influenza.
Currently, the world is making tremendous efforts for the development of antiviral drugs. Among the agents authorized on the market are lamivudine for the treatment of HIV-1 and hepatitis B, ganciclovir for the treatment of herpes virus infections, ribavirin, which is used primarily for the treatment of respiratory syncytial virus and infections and can be used in the treatment of various viral infections in the emergency, amantadine and its analog rimantadine for the treatment of influenza A virus, zanamivir (Relenza) and oseltamivir (TAMIFLU) which are artificially synthesized as neuraminidase inhibitors of influenza virusTM). However, zanamivir has limited routes of administration by inhalation and intravenous injection, and oseltamivir can be administered orally, while its resistant virus has been recently reported, and oral administration has side effects such as vomiting, dizziness, and the like (Ward P et al, j.anitimicrob. chemicother, 55(supp1), p.i. 5-i21,2005). Therefore, there is a need to develop non-toxic side-effect antiviral agents with non-toxic natural ingredients and such vaccines and therapeutics.
In this context, the present inventors have studied to develop a therapeutic agent derived from natural substances for preventing and treating diseases caused by, for example, vesicular stomatitis virus, newcastle disease virus, herpes virus, respiratory syncytial virus and rhinovirus, and successfully confirmed that epimedium koreanum extract, which is a korean traditional herbal medicine, increases the survival rate of cells infected with the above viruses, reduces the number of viruses in the cells, and inhibits intracellular viral replication. As a result, the present inventors have completed the present invention by showing that the korean epimedium brevicornum extract can be effectively used as an active ingredient of an antiviral composition against vesicular stomatitis virus, newcastle disease virus, herpes virus, respiratory syncytial virus and rhinovirus.
Disclosure of Invention
[ problem ] to
The present invention is directed to provide a pharmaceutical composition for preventing and treating viral diseases, which comprises epimedium koreanum extract as an active ingredient.
[ solution ]
In order to achieve the above objects, the present invention provides a pharmaceutical composition for preventing and treating viruses, which comprises epimedium koreanum extract as an active ingredient.
In addition, the present invention provides a functional health food for preventing and alleviating viruses, which comprises epimedium koreanum extract as an active ingredient.
In addition, the present invention provides a pharmaceutical composition for preventing and treating viral diseases, which comprises the epimedium koreanum extract as an active ingredient.
[ advantageous effects ]
The present invention relates to a pharmaceutical composition for preventing and treating viral diseases, which comprises an extract of epimedium koreanum nakai as an active ingredient. The epimedium koreanum extract of the present invention has been confirmed to increase the survival rate of cells infected with the above viruses, reduce the number of viruses in the cells, and inhibit intracellular viral replication, and thus can be effectively used as a functional health food for preventing and alleviating diseases caused by vesicular stomatitis virus, newcastle disease virus, herpes virus, respiratory syncytial virus, and rhinovirus.
Drawings
FIG. 1 shows fluorescence microscopy observations of Green Fluorescent Protein (GFP) displayed during replication of Vesicular Stomatitis Virus (VSV) in virus-infected cells following treatment with the extract of Epimedium koreanum in Korea:
culture medium: a culture medium;
IFN- β/VSV-GFP: cells treated with IFN- β as a positive control after infection with vesicular stomatitis virus attached with Green Fluorescent Protein (GFP);
VSV-GFP: vesicular stomatitis virus with attached green fluorescent protein; and
EYK/VSV-GFP: wherein cells treated with a vesicular stomatitis virus having green fluorescent protein attached thereto are treated with a Korean Medusa herb extract.
FIG. 2 shows cell survival and viral content or number in vesicular stomatitis virus infected cells after treatment with the extract of Epimedium koreanum nakai:
culture medium: a culture medium;
IFN- β/VSV-GFP: cells treated with IFN- β as a positive control after infection with vesicular stomatitis virus attached with Green Fluorescent Protein (GFP);
VSV-GFP: vesicular stomatitis virus with attached green fluorescent protein;
EYK/VSV-GFP: wherein cells treated with a vesicular stomatitis virus having attached green fluorescent protein are treated with a Korean Medusa chinensis extract; and
*: the experiment was performed three times independently, and the case where the p-value of t-test analysis of students was less than 0.05 was determined to be statistically significant.
FIG. 3 shows fluorescence microscopy observations of green fluorescent protein displayed during Newcastle Disease Virus (NDV) replication in virus-infected cells after treatment with Epimedium koreanum extract:
culture medium: a culture medium;
IFN- β/NDV-GFP: cells treated with IFN- β as a positive control after infection with newcastle disease virus with Green Fluorescent Protein (GFP) attached;
NDV-GFP: newcastle disease virus with attached green fluorescent protein; and
EYK/NDV-GFP: wherein the Newcastle disease virus to which green fluorescent protein is attached is treated with an extract of Epimedium koreanum.
FIG. 4 shows the cell survival rate in cells infected with Newcastle disease virus after treatment with the extract of Epimedium koreanum:
culture medium: a culture medium;
IFN- β/NDV-GFP: cells treated with IFN- β as a positive control after infection with newcastle disease virus attached with green fluorescent protein;
NDV-GFP: newcastle disease virus with attached green fluorescent protein;
EYK/NDV-GFP: wherein the cells treated with the Newcastle disease virus to which green fluorescent protein is attached are treated with the Korean Medusa chinensis extract; and
*: the experiment was performed three times independently, and the case where the p-value of t-test analysis of students was less than 0.05 was determined to be statistically significant.
FIG. 5 shows the intracellular viral infection status obtained by hourly measurement of viral matrix mRNA of cells infected with Newcastle disease virus after treatment with Epimedium koreanum extract:
NDV-GFP: newcastle disease virus with attached green fluorescent protein;
EYK/NDV-GFP: wherein the Newcastle disease virus to which green fluorescent protein is attached is treated with an extract of Epimedium koreanum.
FIG. 6 shows fluorescence microscopy observations of green fluorescent protein displayed during replication of herpes virus (HSV) in virus-infected cells after treatment with Epimedium koreanum extract:
culture medium: a culture medium;
IFN- β/HSV-GFP: cells treated with IFN- β as a positive control after infection with a herpes virus with attached Green Fluorescent Protein (GFP);
HSV-GFP: herpes virus with attached green fluorescent protein; and
EYK/HSV-GFP: wherein the herpesvirus having green fluorescent protein attached thereto is treated with an extract of Epimedium koreanum.
FIG. 7 shows the cell survival rate in cells infected with herpes virus after treatment with the extract of Epimedium koreanum:
culture medium: a culture medium;
IFN- β/HSV-GFP: cells treated with IFN- β as a positive control after infection with a herpes virus having green fluorescent protein attached thereto;
HSV-GFP: herpes virus with attached green fluorescent protein;
EYK/HSV-GFP: wherein the herpesvirus attached with green fluorescent protein is treated with Epimedium koreanum extract; and
*: the experiment was performed three times independently, and the case where the p-value of t-test analysis of students was less than 0.05 was determined to be statistically significant.
FIG. 8 shows cell survival rates in rhinovirus-infected cells after treatment with the extract of Epimedium koreanum:
culture medium: a medium containing HeLa cells;
HRV only: HeLa cells infected with rhinovirus;
EYK-0.2%/HRV: treating rhinovirus infected HeLa cells with 0.2% epimedium koreanum extract;
EYK-0.5%/HRV: treating rhinovirus infected HeLa cells with 0.5% epimedium koreanum extract;
EYK-1.0%/HRV: treating rhinovirus infected HeLa cells with 1.0% epimedium koreanum extract;
EYK-2.0%/HRV: HeLa cells infected with rhinovirus were treated with 2.0% of the extract of Epimedium koreanum.
FIG. 9 shows the virus content or amount in rhinovirus infected cells after treatment with the extract of Epimedium koreanum:
culture medium: a medium containing HeLa cells;
HRV only: HeLa cells infected with rhinovirus;
EYK-0.2%/HRV: treating rhinovirus infected HeLa cells with 0.2% epimedium koreanum extract;
EYK-0.5%/HRV: treating rhinovirus infected HeLa cells with 0.5% epimedium koreanum extract;
EYK-1.0%/HRV: treating rhinovirus infected HeLa cells with 1.0% epimedium koreanum extract;
EYK-2.0%/HRV: HeLa cells infected with rhinovirus were treated with 2.0% of the extract of Epimedium koreanum.
Detailed Description
Hereinafter, the present invention will be described in more detail.
The present invention provides a pharmaceutical composition for preventing and treating viruses, which comprises an extract of epimedium koreanum nakai as an active ingredient.
The virus is preferably any one selected from the group consisting of Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV), herpes virus (HSV), Respiratory Syncytial Virus (RSV), and rhinovirus, but is not limited thereto.
The extract is preferably obtained by extracting with water, C1And C2Or a solvent mixture thereof, and the lower alcohol is preferably methanol or ethanol.
In a specific embodiment of the present invention, the present inventors observed the replication of vesicular stomatitis virus using a fluorescence microscope to confirm the effect of epimedium koreanum extract on the inhibition of vesicular stomatitis virus infection. As a result, it was confirmed that the cells infected with vesicular stomatitis virus after treatment with the epimedium koreanum extract showed reduced fluorescence during replication (see fig. 1). In addition, when the present inventors performed trypan blue staining experiments to determine cell viability and the number of viruses in cells, it was confirmed that cells infected with vesicular stomatitis virus after treatment with the epimedium koreanum extract exhibited increased cell viability and decreased number of viruses in cells (see fig. 2).
In addition, the present inventors observed the replication of newcastle disease virus by fluorescence microscopy to confirm the effect of the epimedium koreanum extract on the inhibition of newcastle disease virus infection. As a result, it was confirmed that the newcastle disease virus-infected cells after treatment with the epimedium koreanum extract showed reduced fluorescence during replication (see fig. 3). Further, when the present inventors performed trypan blue assay to determine cell viability and RT-PCR assay to measure mRNA level of virus (matrix) within cells, it was confirmed that cell viability of newcastle disease virus infected cells after treatment with epimedium koreanum extract was increased (see fig. 4) and expression of matrix mRNA was decreased in treated cells (see fig. 5) compared to untreated infected cells.
In addition, the present inventors observed the replication of herpesvirus by fluorescence microscope to confirm the effect of epimedium koreanum extract on inhibition of herpesvirus infection. As a result, it was confirmed that the survival rate of the cells infected with herpes virus after treatment with the epimedium koreanum extract was increased compared to the untreated infected cells (see fig. 6 and 7).
In addition, in order to confirm the effect of the epimedium koreanum extract of the present invention on inhibition of rhinovirus infection, the present inventors pretreated HeLa cell line of epithelial cells with the extracts at concentrations of 0.2%, 0.5%, 1% and 2%, infected it with rhinovirus at 1MOI, and measured cytotoxicity caused by rhinovirus after 24 hours. As a result, it was confirmed that the cell viability depends on the concentration of the extract, increases with increasing concentration, and the virus content or amount decreases (see fig. 8 and 9).
Therefore, the epimedium koreanum extract of the present invention was confirmed to increase the survival rate of cells infected with vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus, and to reduce the virus content or amount in the cells. Since the extract further inhibits intracellular viral replication, it can be effectively used as a pharmaceutical composition for preventing and treating viral diseases caused by vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus.
The compositions of the present invention may also comprise suitable carriers, excipients or diluents conventionally used in the preparation of pharmaceutical compositions.
The compositions of the present invention may be administered orally or parenterally. The parenteral administration thereof is preferably selected from the group consisting of external application to the skin and intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular, and intrapleural injection, but is not limited thereto.
The composition of the present invention can be used as oral preparations (e.g., powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc.), external preparations, suppositories, and sterile injection solutions according to various conventional methods. The carrier, excipient or diluent that may be included in the composition may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The formulations are prepared by using commonly used diluents or excipients (e.g., fillers, extenders, binders, wetting and disintegrating agents, surfactants). Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and are prepared by mixing at least one excipient (e.g., starch, calcium carbonate, sucrose or lactose and a mixture of gelatin with digitalis, saponin, ginseng radix and honey). In addition to these excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration correspond to suspensions, stock solutions, emulsions, syrups and the like. The formulations may contain excipients such as wetting agents, sweeteners, flavours and preservatives, and water and liquid paraffin as common simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), and the like. Suppository bases may include witepsol, polyethylene glycol, tween 61, cocoa butter, bay oil, glycerogelatin, and the like.
Although the preferred administration dose of the composition of the present invention depends on the state, body weight, severity of disease, pharmaceutical dosage form, and administration route and time of the patient, those skilled in the art can appropriately select such a dose. However, for the desired effect, the composition may be administered at a dose of 0.0001g/kg to 1g/kg per day, preferably at a dose of 0.001mg/kg to 200mg/kg, but the dose is not limited thereto. Administration may be once or several times a day. The dosage does not limit the scope of the invention in any way.
In addition, the present invention provides a functional health food for preventing and alleviating viruses, which comprises the extract of epimedium koreanum nakai as an active ingredient.
The virus is preferably selected from any one of Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV), herpes virus (HSV), Respiratory Syncytial Virus (RSV), and rhinovirus, but is not limited thereto.
The extract is preferably obtained by extracting with water, C1And C2Or a solvent mixture thereof, the lower alcohol is preferably methanol or ethanol.
Therefore, the epimedium extract of the present invention was confirmed to increase the survival rate of cells infected with vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus, and to reduce the virus content or amount in the cells. Since the extract further inhibits intracellular viral replication, it can be effectively used as a functional health food for preventing and alleviating viral diseases caused by vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus.
In order to have the above antiviral use, the extract of the present invention may be prepared in various manners disclosed in topical or pharmaceutical and may be any orally ingestible food type as such or in admixture with edible carriers, excipients, diluents and the like. Preferably, the extract is in the form of a beverage, pill, granule, tablet or capsule.
The functional health food of the present invention may further comprise ingredients that are acceptable and conventionally added to foods at the time of preparing the foods. For example, in the preparation of a beverage, at least one component derived from citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and the like is contained in addition to the mixed extract of the present invention.
The amount of the active ingredient that can be included in the functional health food of the present invention can be appropriately selected according to the age, sex, weight, condition, and disease symptoms of the person for whom the functional health food is intended to be used as an antiviral. Preferably, the amount per day of the adult is 0.01g to 10.0g, and the antiviral effect can be produced by ingesting the functional health food in this amount.
In addition, the present invention provides a pharmaceutical composition for preventing and treating viral diseases, which comprises epimedium koreanum extract as an active ingredient.
The viral disease is preferably any one selected from the group consisting of vesicular stomatitis, newcastle disease, hand-foot-and-mouth disease, meningitis, influenza, bronchitis, pneumonia, bronchiolitis and acute hepatitis, but is not limited thereto.
Therefore, the epimedium koreanum extract of the present invention was confirmed to increase the survival rate of cells infected with vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus, and to reduce the virus content or amount in the cells. Since the extract further inhibits intracellular viral replication, it can be used as a pharmaceutical composition for preventing and treating viral diseases caused by vesicular stomatitis virus, newcastle disease virus, herpes virus and rhinovirus.
Examples
The present invention will be described in more detail below with reference to examples.
However, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed to limit the scope of the present invention.
< example 1> preparation of Epimedium koreanum extract
Epimedium koreanum from korea was purchased and used as a sample to prepare an extract of epimedium koreanum. 1L of water was added to about 50g of carefully selected Epimedium koreanum, and hot water extraction was performed at 115 ℃ for 3 hours using a medical hot plate (Gyeong seo Extractor Cosmos-600, Nakawa, Korea) to obtain about 100mL of Epimedium koreanum extract. The extract was filtered using standard test sieves (150 μm; Leichi, Haen, Germany) and stored at 4 ℃ until used in the experiment.
< example 2> cell culture
For antiviral efficacy experiments of Epimedium koreanum extract, murine macrophage cell line RAW 264.7 cells and human embryonic kidney cell line 293T were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen Life technologies) supplemented with 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and 100 μ g/ml streptomycin, respectively, at 37 deg.C in the presence of 5% CO2The humidity of the incubator is 95%. Cells were subcultured with fresh medium every 2 or 3 days.
< example 3> confirmation of inhibitory Effect on Vesicular Stomatitis Virus (VSV) infection
<3-1> confirmation of inhibitory Effect on viral infection Using fluorescence microscope
To confirm the inhibitory effect of the epimedium koreanum extract on vesicular stomatitis virus infection, the replication of the virus was observed under a fluorescent microscope as described below.
Specifically, RAW 264.7 cell line was treated with 1% of the epimedium koreanum extract prepared in < example 1> above. After 12 hours, the cell line was infected with 1MOI of vesicular stomatitis virus (VSV-gfp virus) to which green fluorescent protein (gfp) was attached, and further cultured for 12 or 24 hours. Thereafter, intracellular replication of VSV-gfp virus was observed by gfp fluorescence. 1000U/ml IFN- β was used as a positive control.
As a result, it was confirmed that the cells infected with vesicular stomatitis virus after the epimedium toward fresh chinese epimedium extract treatment showed reduced fluorescence during replication (see fig. 1).
<3-2> confirmation of inhibitory Effect against viral infection Using Trypan blue staining test
To confirm the inhibitory effect of epimedium koreanum extract on vesicular stomatitis virus infection, trypan blue staining test was performed to determine the cell viability of cells and the number of viruses in cells.
Specifically, the medium was removed from the cells cultured in the same manner as in example <3-1> above, and the cells were treated with 0.05% trypsin-EDTA and floated. Then, an appropriate amount of Phosphate Buffered Saline (PBS) was added to each well, and the cells were agglutinated and centrifuged at 2000rpm for 5 minutes. When the cells were left by themselves after removing the supernatant, the cells were mixed well with 1mL of PBS added, and further mixed with an equal amount of cell suspension and 0.5% trypan blue (Gibco BRL). The mixture was treated for 2 minutes and viable cells were counted under a fluorescent microscope. 1000U/ml IFN- β was used as a positive control. Cell viability is described as a percentage on the culture medium. All experimental results were of three independent experiments, P <0.05 indicating significant differences between the comparative groups by student t-test.
As a result, as shown in fig. 2, it was confirmed that the cells infected with vesicular stomatitis virus after treatment with the epimedium koreanum extract exhibited increased cell survival rate (see fig. 2A) and decreased the number of viruses in the cells (see fig. 2B). All experimental results were of three independently performed experiments, P <0.05 indicating significant differences between the comparative groups by student t-test.
< example 4> confirmation of inhibitory Effect on Newcastle Disease Virus (NDV) infection
<4-1> confirmation of inhibitory Effect on viral infection Using fluorescence microscope
In order to confirm the inhibitory effect of the epimedium koreanum extract on the newcastle disease virus infection, the replication of the virus was observed under a fluorescent microscope in the same manner as in the above example <3-1 >.
As a result, it was confirmed that the cells infected with newcastle disease virus after the treatment of the epimedium toward xianzhui extract showed reduced fluorescence during replication (see fig. 3).
<4-2> confirmation of inhibitory Effect on viral infection Using Trypan blue staining test and reverse transcription-polymerase chain reaction (RT-PCR)
To confirm the inhibitory effect of epimedium koreanum extract on newcastle disease virus infection, trypan blue staining test was performed to determine cell viability, and viral matrix mRNA was measured using RT-PCR assay to count intracellular viruses.
In particular, in accordance with the embodiments described above<3-2>The same procedure was used for trypan blue staining. For RT-PCR assays, the above examples will be described<3-1>The cells cultured in the method described in (1) were washed with PBS stored at 4 ℃ and separated using a spatula. The separated cells were centrifuged at 1200rpm and 4 ℃ for 2 minutes, and after removing the supernatant, a precipitate was obtained. Total RNA was then extracted using the RNeasy Plus Mini kit (Qiagen, Valencia, CA) under the manufacturer's protocol. DEPC-treated water was used to dissolve RNA and the absorbance of the RNA was measured using an a260/280 ratio (Versa Max microplate reader, molecular devices) to identify purity and concentration. The amount of transcribed mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Using oligo- (dt)15The primers synthesized the same amount of RNA as cDNA and PCR was performed using Power SYBR Green PCR Master Mix reagent (applied biosystems) and 7500Fast Real time-PCR system (applied biosystems). PCR was performed at 50 ℃ for 20 seconds and 95 ℃ for 10 minutes, andthe relative band intensities of the proton (M) protein and GAPDH are shown in the figure. The primers used in the above RT-PCR are disclosed in Table 1 below.
As a result, as shown in fig. 4 and 5, it was confirmed that the survival rate of the cells infected with newcastle disease virus after the epimedium toward fresh chinese epimedium extract treatment was increased (see fig. 4) and the expression of matrix mRNA was decreased in the treated cells (see fig. 5) compared to the untreated infected cells. All experimental results were of three independent experiments, P <0.05 indicating significant differences between the comparative groups by student t-test.
[ Table 1]
Figure BDA0001268197180000141
< example 5> confirmation of inhibitory Effect on herpes Virus (HSV) infection
In order to confirm the effect of epimedium koreanum extract on inhibition of herpes virus infection, the following experiment was performed.
Specifically, 1% of the epimedium koreanum extract prepared in < example 1> above was treated on the 293T cell line for 12 or 24 hours, the cell line was infected with 2MOI of herpes virus (HSV-GFP), and cytotoxicity of the herpes virus was measured. Specifically, 1000U/ml IFN- β was used as a positive control. The cell line was observed under a microscope, and the viability of the cultured cells was determined by trypan blue staining test in the same manner as in example <3-2> above.
As a result, as shown in fig. 6 and 7, it was confirmed that the survival rate of the cells infected with herpes virus after the treatment with the epimedium koreanum extract was increased compared to the untreated infected cells (see fig. 6 and 7). All experimental results were of three independently performed experiments, P <0.05 indicating significant differences between the comparative groups by student t-test. Thus, the inhibitory effect of epimedium koreanum on herpes virus infection as a DNA virus was confirmed.
< example 6> confirmation of inhibitory Effect on rhinovirus (HSV) infection
In order to confirm the inhibitory effect of the epimedium koreanum extract on rhinovirus infection, the following experiment was performed.
Specifically, 0.2%, 0.5%, 1% and 2% of the epimedium koreanum extract prepared in < example 1> above was treated on HeLa cell line of epithelial cells, the cell line was infected with rhinovirus at 1MOI, and cytotoxicity of rhinovirus was measured after 24 hours. The cell line was observed under a microscope, and the viability of the cultured cells was determined by the trypan blue staining test in the same manner as in example <3-2> above.
As a result, it was confirmed that the cell viability depends on the concentration of the extract, increases with increasing concentration, and the virus content or amount decreases (see fig. 8 and 9).
<110> Korean institute of medicine
<120> antiviral composition comprising extract of epimedium koreanum nakai as active ingredient
<130> OPA15219
<150> KR10-2014-0104220
<151> 2014-08-12
<150> KR10-2014-0135163
<151> 2014-10-07
<160> 4
<170> KopatentIn 3.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> APMV-1M _ F primer
<400> 1
agtgatgtgc tcggaccttc 20
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> APMV-1M _ R primer
<400> 2
cctgaggaga ggcatttgct a 21
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> GAPDH _ F primer
<400> 3
tgaccacagt ccatgccatc 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> GAPDH _ R primer
<400> 4
gacggacaca ttgggggtag 20

Claims (8)

1. Use of an extract of Epimedium koreanum nakai in the manufacture of a medicament for the prevention and treatment of viral diseases, wherein the virus is Vesicular Stomatitis Virus (VSV).
2. Use according to claim 1, wherein the extract is obtained by mixing water, C1And C2Or a solvent mixture thereof.
3. The use according to claim 1, wherein the lower alcohol is methanol or ethanol.
4. The use of claim 1, wherein the viral disease is vesicular stomatitis.
5. Use of an extract of Epimedium koreanum nakai in the manufacture of a medicament for the prevention and treatment of viral diseases, wherein the virus is Vesicular Stomatitis Virus (VSV) and at least one selected from the group consisting of herpes virus (HSV), Respiratory Syncytial Virus (RSV) and rhinovirus.
6. Use according to claim 5, wherein the extract is obtained by mixing water, C1And C2Or a solvent mixture thereof.
7. Use according to claim 5, wherein the lower alcohol is methanol or ethanol.
8. The use of claim 5, wherein the viral disease is vesicular stomatitis, and at least one selected from the group consisting of bronchitis, pneumonia, and bronchiolitis.
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