KR20230076953A - Antiviral composition comprising extract of Oenothera biennis L. - Google Patents

Abstract

The present invention relates to an antiviral composition containing an extract of Oenothera biennis, and specifically relates to the use of an extract of Oenothera biennis for preventing, alleviating or treating coronavirus infection. The extract of Oenothera biennis of the present invention exhibits an effect of suppressing coronavirus infection, it can be provided for antiviral purposes.

Description

Antiviral composition comprising extract of Oenothera biennis L.}

The present invention relates to an antiviral composition comprising an extract of Oenothera biennis L. , and specifically to a use for preventing, ameliorating or treating a coronavirus infection comprising an extract of Oenothera biennis L.

Viruses continue to threaten human health, and in particular, new and mutated viruses are frequently appearing. An aging population, an increase in immunocompromised persons, and an increase in international trade and travel are factors contributing to the acceleration of virus transmission. For example, it has been estimated that 4.0-8.8 people per 100,000 people die from influenza-related respiratory diseases annually due to seasonal influenza virus epidemics. Coronavirus Disease-19 (COVID-19), which started in Wuhan, China in 2019, has caused more than 256 million patients and more than 5.13 million deaths worldwide as of mid-November 2021 It became.

Among viruses, coronavirus refers to viruses belonging to the Coronaviridae family having single-stranded RNA as a genome, and is found in various mammals including humans as well as birds. Coronaviruses are known to cause both respiratory and digestive infectious diseases depending on the characteristics of the virus and its host.

The coronavirus is a pathogen of not only the aforementioned COVID-19, but also of Severe Acute Respiratory Syndrom (SARS) in 2003 and Middle East Respiratory Syndrome Coronavirus (MERS) in 2015. New and mutated viruses are continuously appearing.

On the other hand, Oenothera biennis L. is also called evening primrose as a plant belonging to the genus Oenothera, and is known to contain γ-linolenic acid, which is rarely found in other plants. It is known to exhibit antihyperlipidemia, antiatherosclerosis, blood pressure lowering, anti-inflammatory, anti-tumor and anti-obesity effects through pharmacological studies.

However, nothing is known about the antiviral effect of moonshine extract against coronavirus.

Under this background, the present inventors completed the present invention by confirming that the moongyeoncho extract exhibits an antiviral effect against coronavirus.

1. Noh Ji-yoon, Antiviral effect on coronavirus.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating viral infections comprising an extract of Oenothera biennis L. as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating a viral infection, comprising the step of treating a subject with a composition containing a moonshine extract.

Another object of the present invention is to provide a food composition for preventing or ameliorating viral infections, containing moonshine extract as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving viral infection, comprising a moonshine extract as an active ingredient.

Each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention is to provide a pharmaceutical composition for preventing or treating viral infections comprising an extract of Oenothera biennis L. as an active ingredient.

The term of the present invention, " Oenothera biennis L. " is also called evening primrose as a plant belonging to the genus Oenothera, and is known to contain γ-linolenic acid, which is rarely found in other plants. It is known to exhibit antihyperlipidemia, antiatherosclerosis, blood pressure lowering, anti-inflammatory, anti-tumor and anti-obesity effects through pharmacological studies.

Moongyeoncho used in the present invention can be collected from nature or purchased commercially, but is not limited thereto.

The term "extract" of the present invention refers to a liquid component obtained by immersing a desired substance in various solvents and then extracting at room temperature, low temperature, or elevated temperature for a certain period of time, and a product such as a solid component obtained by removing the solvent from the liquid component. means In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.

In the present invention, the extract may be a moongyeoncho extract. The extract can be extracted from various organs of natural, hybrid, and mutant plants of the target plant, for example, roots, aerial parts, stems, leaves, flowers, fruit bodies, and fruit skins as well as plant tissues, It may be appropriately selected according to the type of target plant, but is not limited thereto. The extract may be obtained by extraction with water or various organic solvents. At this time, the organic solvent used is not particularly limited thereto as long as the extract can be obtained, but specifically, it may be water, a polar solvent or a non-polar solvent, and more specifically, water, a lower alcohol having 1 to 4 carbon atoms ( As a C1 to C4 alcohol, it may be any one or more selected from methanol or ethanol, etc.) and mixed solvents thereof.

In addition, the method for obtaining the extract is not particularly limited thereto, as long as the extract can be obtained, but specifically, the root, stem, leaf, fruit, flower, dried product, processed product, etc. of the target plant Methods such as immersion in a solvent and extraction at room temperature, cold extraction, heating extraction, ultrasonic extraction by applying ultrasonic waves, and reflux extraction using a reflux condenser may be used.

Alternatively, commercially available moongyeoncho extract may be purchased and used.

In the present invention, it was confirmed through a specific experiment that the wolgyeoncho extract inhibits viral infection, and it was first identified that it can be used for prevention, improvement, or treatment of viral infection.

In the present invention, the virus may be a coronavirus, but is not limited thereto.

The coronavirus is, for example, SARS-CoV-2, a novel coronavirus discovered in 2019, Severe Acute Respiratory Syndrome (SARS)-causing coronavirus (SARS-CoV) discovered in 2003, 2012 Severe Acute Respiratory Syndrome (SARS)-causing coronavirus (MERS-CoV), also known as community-acquired respiratory HCoV (CAR HCoV), HCoV-229E, HCoV-OC43 , HCoV-NL63 and HKU1, but not limited thereto.

In the present invention, the term "prevention" refers to any activity that suppresses or delays the onset of a viral infection by administration of the composition, and "treatment" means that symptoms caused by a viral infection are improved or beneficially changed by administration of the composition. means any action that

The pharmaceutical composition of the present invention may further include suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions. At this time, the content of the active ingredient included in the pharmaceutical composition is not particularly limited thereto, but may include 0.0001% to 10% by weight, specifically 0.001% to 1% by weight based on the total weight of the composition.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. It may have one dosage form, and may be various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The composition of the present invention may be for oral administration, but is not limited thereto.

The composition of the present invention can be administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, and disease. It may be determined according to factors including type, activity of drug, sensitivity to drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administration. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route of administration and the period, but for a desired effect, the composition of the present invention is 0.0001 to 100 mg / kg per day, specifically As such, it is preferable to administer 0.001 to 10 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The composition can be administered to various mammals such as rats, livestock, and humans by various routes, and the method of administration includes without limitation as long as it is a conventional method in the art, for example, oral, rectal or intravenous, intramuscular, It may be administered by subcutaneous, intrauterine, or intracerebrovascular injection. Specifically, it may be oral administration, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention can be used in the form of animal medicine as well as medicine applied to humans. Here, the animal is a concept including livestock and companion animals.

Another aspect of the present invention is to provide a method for preventing or treating a viral infection, comprising the step of treating a subject with a composition containing a moonshine extract.

Terms used herein are as described above.

In the present invention, the term "subject" refers to all animals other than humans that have or may have a viral infection, and by administering the pharmaceutical composition of the present invention to a subject suspected of having a viral infection, the subject can be efficiently treated.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject suspected of having a viral infection by any appropriate method, and the route of administration is through various oral or parenteral routes as long as it can reach the target tissue. can be administered.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, as described above.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of preventing or treating a viral infection, and is applicable to any individual. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, and fish may be used, and the pharmaceutical composition may be used parenterally, subcutaneously, or intraperitoneally. It can be administered internally, intrapulmonaryly and intranasally, and for local treatment, it can be administered by any suitable method, including intralesional administration if necessary. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the subject, the severity of the disease, the drug type, the route of administration and the period, but can be appropriately selected by those skilled in the art. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection, but is not limited thereto.

In the present invention, the virus may be a coronavirus, but is not limited thereto.

Another aspect of the present invention is to provide a food composition for preventing or ameliorating viral infection, comprising a moonshine extract as an active ingredient.

Terms used herein are as described above.

In the present invention, the term "improvement" refers to all activities that improve or benefit the symptoms of suspected or affected individuals of viral infections by using the composition.

[0025] Food-wise acceptable salts that can be included in the food composition of the present invention are useful acid addition salts formed from food-acceptable free acids or metal salts formed from bases. As an example, inorganic acids and organic acids may be used as free acids. Hydrochloric acid, sulfuric acid, hydrobromic acid, sulfurous acid, or phosphoric acid may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, or the like may be used as the organic acid. In addition, as the metal salt, an alkali metal salt or an alkaline earth metal salt, sodium, potassium or calcium salt may be used. However, it is not limited thereto.

The food composition of the present invention includes forms such as pills, powders, granules, precipitates, tablets, capsules or liquids, and the food to which the composition can be added includes, for example, various foods, such as beverages, There are chewing gum, tea, vitamin complexes, and health supplements.

Ingredients that can be included in the food composition of the present invention are not particularly limited to other ingredients other than containing active ingredients as essential ingredients, and, like conventional foods, various herbal extracts, food supplement additives, or natural carbohydrates as additional ingredients. may contain The content of the active ingredient in the food composition may be appropriately determined depending on the purpose of use (prevention, improvement or therapeutic treatment). At this time, the content of the active ingredient included in the composition is not particularly limited thereto, but may include 0.0001% to 10% by weight, specifically 0.001% to 1% by weight based on the total weight of the composition.

In addition, the food auxiliary additives may include food auxiliary additives common in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.

Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above, natural flavors (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can advantageously be used as flavoring agents.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, it may contain fruit flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

In the present invention, the health supplement may include health functional food and health food.

The health functional food (functional food) is the same term as food for special health use (FoSHU), and has high medical effect and is processed to efficiently display bioregulatory functions in addition to nutrient supply. means food. Here, "function (sex)" means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions.

Food containing the food composition of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and has excellent portability.

In the present invention, the virus may be a coronavirus, but is not limited thereto.

Another aspect of the present invention is to provide a feed composition for preventing or ameliorating viral infection, comprising a moonshine extract as an active ingredient.

Terms used herein are as described above.

The feed composition may be in the form of a feed additive composition in addition to the feed composition.

When the composition is prepared as a feed additive, the composition may be 20 to 90% high concentration or may be prepared in powder or granular form. The feed additives are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, and rosemary. extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like, or any one or more natural antioxidants may be further included. When prepared as a feed, the composition may be formulated in a conventional feed form, and may include common feed ingredients together.

The feed and feed additives include grains such as milled or ground wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean, and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; It may further include sugar and dairy products, for example, dry ingredients composed of various powdered milk and whey powder, etc., and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered to animals as a top dressing, directly mixed with animal feed, or in an oral formulation separate from feed. When the feed additive is administered separately from animal feed, it can be prepared as an immediate release or sustained release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution formulation.

In addition, the feed and feed additives may contain auxiliary agents, such as preservatives, stabilizers, wetting agents or emulsifying agents, solution accelerators, and the like. The feed additive may be used by adding it to an animal's feed by steeping, spraying or mixing.

The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish.

As the mammal, it can be used for pigs, cows, horses, sheep, rabbits, goats, rats, hamsters, and guinea pigs, which are rodents and laboratory rodents, as well as companion animals (eg, dogs and cats), and as the poultry, chickens, It can also be used for turkeys, ducks, geese, pheasants, and quails, and can be used for carp, crucian carp, and trout as the fish, but is not limited thereto.

In the present invention, the virus may be a coronavirus, but is not limited thereto.

Since the moongyeoncho extract of the present invention exhibits a coronavirus infection inhibitory effect, it can be provided for antiviral use.

1 is a diagram showing the inhibitory effect of HCoV-OC43 and HCoV-229E infection on MRC-5 cells of moonshine extract.
Figure 2 is a view showing the SARS-CoV-2 infection inhibitory effect on Vero cells of the moongyeoncho extract.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited only to these examples.

Example 1: Preparation of Moongyeoncho extract

Moongyeoncho ( Oenothera biennis L. ) was extracted under reflux in 70% ethanol about 10 times its weight for 3 hours, filtered through a sieve and freeze-dried to prepare an ethanol extract of moongyeoncho in powder form. For use in the experiment, it was dissolved in 50% DMSO at a concentration of 50 mg/ml, filtered through a 0.22 μm syringe filter, and stored frozen at -20°C.

Example 2: Evaluation of cell infection inhibition activity of HCoV by moonshine extract (Co-treatment)

Check whether the moonshine extract prepared in Example 1 inhibits the infection of HCoV-OC43 and HCoV-229E belonging to community-acquired respiratory HCoV (CAR HCoV), a coronavirus that infects humans. In order to do this, the cell-based in vitro antiviral efficacy was evaluated by a co-treatment method. The antiviral efficacy of the extract was determined by a dose response curve (DRC) experiment in a cell infection model of HCoV.

Specifically, 2 Х 10 ⁵ MRC-5 cells were inoculated per well in a 24-tissue culture plate and cultured for 24 hours. After reacting each virus (HCoV-OC43, 0.01 MOI; HCoV-229E, 0.5 MOI) and moonshine extract (0.1, 1, 10 μg/mL) at 4°C for 1 hour, the virus and extract were added to the cell culture medium. The reaction solution was added and infected for 2 hours at 37°C. Thereafter, the reaction solution was removed, replaced with a new culture solution, and then incubated at 37° C. for 72 hours, and the cell infection inhibition rate was calculated through MTT assay.

As a result, the moonshine extract inhibited HCoV-OC43 and HCoV-229E infection of MRC-5 cells in a concentration-dependent manner (FIG. 1).

Example 3: Evaluation of cell infection inhibition activity of SARS-CoV-2 by moonshine extract

Cell-based in vitro antiviral efficacy was evaluated to determine whether the moonshine extract prepared in Example 1 inhibits the infection of SARS-CoV-2 (Wuhan Coronavirus-19, provided by the Centers for Disease Control and Prevention). The antiviral efficacy of the extract was determined by DRC experiments in a cell infection model of SARS-CoV-2.

Specifically, 1.2 Х 10 ⁴ Vero cells (ATCC-CCL81) were inoculated per well in a 384-tissue culture plate, and 24 hours later, a 2-fold serially diluted sample of the moonshine extract was treated with the cells for about 1 hour. Then SARS-CoV-2 (0.0125 MOI) was infected and cultured for 24 hours. Thereafter, the cells were fixed with 4% PFA (paraformaldehyde), permeabilized, and treated with anti-SARS-CoV-2 nucleocapsid (N) primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. , Hoechst 33342 was treated to stain the cells.

Fluorescence images of infected cells were acquired using Operetta (Perkin Elmer) and analyzed using Columbus software.

The total number of cells per well was calculated as the number of Hoechst-stained nuclei, and the number of infected cells was calculated as the number of cells expressing the viral N protein. Infection ratio was calculated as the number of cells expressing N protein/total number of cells.

The infectivity per well was normalized to the average infectivity of wells containing uninfected cells (mock) and the average infectivity of wells containing infected cells treated with 0.5% DMSO (Dimethyl Sulfoxide) (v/v) in the same plate.

In addition, the cell viability of the extract was indicated as 'cell viability' in the graph by normalizing the number of cells in each well with the average number of cells in the mock group wells.

As a result, the moonshine extract inhibited SARS-CoV-2 infection of Vero cells while showing little cytotoxicity (FIG. 2).

The results of the above examples indicate that it has the effect of inhibiting coronavirus infection in cells and is safe and has no side effects because it does not exhibit cytotoxicity, indicating that the moonshine extract can be used for the prevention, improvement or treatment of viral infections. it suggests

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (7)

Moongyeoncho ( Oenothera biennis L. ) Containing an extract as an active ingredient, a pharmaceutical composition for preventing or treating viral infections.
The composition according to claim 1, wherein the extract is extracted with at least one selected from the group consisting of water, lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.
The composition of claim 1, wherein the virus is a coronavirus.
The composition of claim 3, wherein the coronavirus is any one or more selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and HKU1. .
A method for preventing or treating a viral infection comprising the step of treating a non-human object with a composition containing an extract of moonshine.
A food composition for preventing or improving viral infection, comprising an extract of moonshine as an active ingredient.
A feed composition for preventing or improving viral infection, comprising an extract of moonshine as an active ingredient.

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