KR102369690B1 - Anti-Coronavirus composition comprising Lonicera praeflorens extract as effective component - Google Patents

Abstract

The present invention relates to an anti-coronavirus composition comprising an extract of Lonicera praeflorens as an active ingredient. It will be very usefully utilized as a composition for prevention, improvement or treatment of diseases caused by

Description

Anti-coronavirus composition comprising Lonicera praeflorens extract as effective component

The present invention relates to a composition for anti-coronaviruses containing an extract of oleracea as an active ingredient.

Respiratory disease, which started in Wuhan, China in 2019, is a disease caused by a coronavirus infection, showing severe respiratory symptoms, causing some of the infected patients to die. To date, approved treatments for coronavirus infection include ribavirin, famciclovir, nitazoxanide, nafamostat, and chloroquine. Vir (remdesivir, GS-5734) and favipiravir are used to treat coronavirus infection, but they are ineffective and have a high incidence of variant viruses due to the nature of nucleic acid analog antiviral agents, so two or more antiviral agents are being applied together ( Wang M. et al., 2020. Cell Research 30:269-271).

Most of the antiviral agents used for coronavirus infection, such as ribavirin and remdesivir, have RNA polymerase inhibitory ability. However, mouse hepatitis virus, a type of coronavirus that has resistance to remdesivir, has been found, and the existing antiviral agents have no effect. In addition, since the antiviral agents developed to date exhibit severe side effects, much attention is required in their application. Therefore, there is an increasing need for the development of excellent new anti-coronavirus agents with excellent infection suppression effect and low toxicity for preventing and treating the outbreak of coronavirus.

Lonicera praeflorens is a deciduous shrub of the Caprifoliaceae family, and is also referred to as a magnolia tree. It grows in mountain forests, young branches have black spots on a brown background, and old branches are gray. The bark is split vertically and the inside of the stem is white. Also, the leaves are opposite, egg-shaped or oval-shaped, with a sharp tip, a round bottom, flat edges, and dense soft hairs on both sides. Flowers are pale red or yellowish-white in March-May, and bloom before leaves. Fruits are berries, ball-shaped, and ripen in red in May-October. The flowers and fruits are edible, and the flowers, leaves, stems, and roots are used medicinally (the crude drug name is ginseng ginseng), and are known to be effective against malaria, bronchitis, and tonsillitis.

Korea Patent No. 0983647 discloses a 'composition for the treatment of hepatitis C' consisting of pumpkin seed, safflower, plantain ( Plantago asiatica L.) and honeysuckle ( Loicera japonica PHUMB), and a Korean registered patent Although 0511514 discloses 'antiviral agents from plant extracts and their use for the treatment of viral infections'. There is no description about the composition for anti-coronavirus containing the extract of the present invention as an active ingredient.

The present invention was derived from the above needs, and while the present inventors were identifying natural (plant) materials for developing novel coronavirus therapeutics, Lonicera praeflorens extract is a type of coronavirus in swine epidemic By confirming that it has an excellent virus proliferation inhibitory effect on diarrhea virus (Porcine epidemic diarrhea virus), infectious gastroenteritis coronavirus (Transmissible gastroenteritis coronavirus) and swine respiratory coronavirus (Porcine respiratory coronavirus), the present invention was completed.

In order to solve the above problems, the present invention provides an anti-coronavirus composition containing an extract of Lonicera praeflorens as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of coronavirus infection containing the extract of oleifera tree as an active ingredient.

In addition, the present invention provides a health functional food composition for the prevention or improvement of coronavirus infection containing the extract of oleifera tree as an active ingredient.

In addition, the present invention provides a feed additive composition for anti-coronavirus containing the extract of olgobula tree as an active ingredient.

In addition, the present invention provides an anti-viral cleaning product containing an extract of oleracea as an active ingredient.

According to the present invention, it can be effectively applied to the prevention and treatment of infectious diseases caused by various viruses, particularly coronaviruses, by using the extract of the mulberry tree having excellent antiviral effect. Since it rarely causes toxicity or side effects, it can be safely used for long-term use for preventive purposes. Therefore, the composition according to the present invention can be applied for the prevention and treatment of infectious diseases caused by viruses, and can be widely used as health functional food, livestock feed additive, cleaning product, and the like.

Figure 1 shows the antiviral activity against the swine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) of olgobulnae extract. AZT: azidothyymidine.
Figure 2 shows the antiviral activity against Transmissible gastroenteritis coronavirus (TGEV) of the olgobulnae extract.
Figure 3 shows the antiviral activity of the extract of olgobulnae against porcine respiratory coronavirus (PRCV).

In order to achieve the object of the present invention, the present invention provides an anti-coronavirus composition containing an extract of Lonicera praeflorens as an active ingredient.

As used herein, the term "anti-coronavirus" means inhibiting the proliferation of coronavirus. Coronaviruses refer to viruses belonging to the family Coronaviridae and are generally found in birds as well as various mammals including humans. Coronaviruses have various species, and are known to cause both respiratory and digestive infectious diseases depending on the characteristics and host of the virus. Coronavirus is a virus with an RNA genome surrounded by an outer membrane and is found among humans, mammals, and birds. The virus was first discovered and reported in the early 1930s in chickens with infectious bronchitis, pigs with infectious gastroenteritis, and mice with severe hepatitis or neurological symptoms. Through an electron microscope, a characteristic shape with a club-shaped protrusion was observed on the surface of the virus particle, and it was named coronavirus because it was derived from the Latin word 'corona', meaning crown, reminiscent of a crown. In the past 40 years, coronavirus has been reported as a causative agent in respiratory and digestive-related infectious diseases that mainly affect livestock economically. Representatively, avian infectious bronchitis virus (Infectious bronchitis virus), swine epidemic diarrhea virus (Porcine epidemic diarrhea virus), feline infectious peritonitis virus (Feline Infectious Peritonitis virus), bovine coronavirus (Bovine coronavirus) and the like are known.

In the anti-coronavirus composition of the present invention, it is preferable to extract the extract of the oleifera tree with one or more solvents selected from water, alcohols having 1 to 4 carbon atoms, and mixtures thereof, more preferably methanol, ethanol or butanol. Extraction using a solvent, and more preferably extraction using ethanol as a solvent may be used. The ethanol extraction of the banyan tree includes the steps of 1) extracting at a temperature of 25 to 60° C. for 68 to 77 hours by adding 5 to 25 times the ethanol based on the dry leaf weight of the banyan tree; 2) sonicating the ethanol extract for 10 to 50 minutes; and 3) centrifuging the sonicated ethanol extract to remove the precipitate; preferably, but not limited thereto.

In addition, the extract obtained from the extract of the oleracea contains any one of an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and a crude product or a purified product.

In the anti-coronavirus composition according to an embodiment of the present invention, the coronavirus is porcine epidemic diarrhea virus, transmissible gastroenteritis coronavirus, porcine respiratory coronavirus, Severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus Virus HKU1 (HCoV-HKU1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), 2019 novel coronavirus (SARS-CoV-2), Canine coronavirus and equine coronavirus It may be any one selected from the group consisting of a virus (Equine coronavirus), preferably a porcine epidemic diarrhea virus, a Transmissible gastroenteritis coronavirus or a swine respiratory coronavirus (Porcine respiratory coronavirus). can, but is not limited thereto.

The extract of the present invention not only has the effect of inhibiting the proliferation of coronavirus, but also exhibits almost no cytotoxicity or side effects, so it can be safely used even when taken for a long period of time.

The present invention also provides a pharmaceutical composition for preventing or treating coronavirus infection containing an extract of Lonicera praeflorens as an active ingredient.

In the pharmaceutical composition for the prevention or treatment of coronavirus infection according to the present invention, the extract of olgobulum is the same as described above.

In addition, the coronavirus infection means a disease caused by infection with a coronavirus, and for example, in humans, it may be a cold, cough, sputum, bronchitis or severe respiratory disease, and in animals, hepatitis, encephalitis, neurological disease, severe respiratory disease. It may be a disease, severe diarrhea or respiratory disease, peritonitis, etc., but is not limited thereto. The type of coronavirus that causes the coronavirus infection is the same as described above.

The term "prevention" used in the present invention refers to any action that suppresses infection of coronavirus or delays the onset of coronavirus infection by the composition of the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of coronavirus infection are improved or beneficially changed by the composition of the present invention.

For administration, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions, respectively, according to conventional methods. . In detail, when formulating, it may be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, and a surfactant commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, Witepsol, Macrosol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. can be used.

The pharmaceutical composition of the present invention may include, with respect to the total weight of the composition, 0.1 ~ 1,000 ㎍ / ㎖, specifically 0.1 ~ 200 ㎍ / ㎖, more specifically 0.1 ~ 100 ㎍ / ㎖ It is not limited thereto.

The method of administration of the pharmaceutical composition is determined according to the severity of symptoms, and a topical administration method is generally preferred. In addition, the effective amount of the active ingredient of the pharmaceutical composition means an amount required for the prevention or treatment of diseases. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health status, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and concomitant drugs. Although not limited thereto, for example, in the case of an adult, generally about 5 to 500 mg/kg, preferably about 100 to 250 mg/kg may be administered 1 to 3 times a day.

It is apparent to those skilled in the art that the formulation method, dosage, route of administration, components, etc. of the pharmaceutical composition of the present invention can be appropriately selected from conventional techniques known in the art.

The pharmaceutical composition of the present invention may be used as an active ingredient in combination with other pharmaceutically active ingredients in addition to the oleifera extract, or mixed with a pharmaceutical composition including other antiviral active ingredients.

The present invention also provides a health functional food composition for preventing or improving coronavirus infection containing an extract of Lonicera praeflorens as an active ingredient.

In the health functional food composition of the present invention, the extract and the coronavirus infection are the same as described above.

As used herein, the term “improvement” refers to any action that reduces a parameter related to a condition to be treated by ingestion of the health functional food composition of the present invention, for example, the degree of symptoms.

As used in the present invention, the term "health functional food" is the same term as food for special health use (FoSHU), and is a medical treatment processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. , refers to foods with high medical effects. Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement may be used interchangeably.

The health functional food composition for the prevention or improvement of coronavirus infection of the present invention is a food prepared by adding or encapsulating, powdering, suspension, etc., to food materials such as beverages, teas, spices, gums, and confectionery , it means that certain health effects are obtained when ingested, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.

The health functional food 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.

In addition, the health functional food composition can be prepared without limitation as long as it is a food-approved formulation in various types of formulations.

In addition, the health functional food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the health functional food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the health functional food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), Butylhydroxytoluene (BHT), etc.), colorant (tar pigment, etc.), color developer (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweetener (dulcin, cyclmate) , saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (foams), film agents, gum base agent, foam inhibitor, solvent, improving agent, etc. of food additives. The additive may be selected according to the type of food and used in an appropriate amount.

The content of the extract of oleracea in the health functional food composition according to the present invention may be appropriately selected in consideration of the shape, flavor, taste, etc. of the food, for example, 0.01 to 30% by weight based on the total weight of the health functional food. can be a range. It is clear to those skilled in the art that the form, composition and manufacturing method of the health functional food composition of the present invention may be appropriately selected from conventional techniques known in the art.

There is no particular limitation on the type of health functional food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes.

The present invention also provides an anti-coronavirus feed additive composition containing an extract of Lonicera praeflorens as an active ingredient.

As used in the present invention, the term "feed additive" is a generic term for substances added in trace amounts to feed for animal nutrition or specific purposes. Substances added to feed for the purpose of Here, an animal is a concept including livestock and pets.

As a component that can be included in the feed additive composition of the present invention, there is no particular limitation on other components other than containing an active ingredient as an essential component. The feed additive composition of the present invention may further include a binder, an emulsifier, a preservative, etc. added to prevent quality deterioration, and an amino acid agent, a vitamin agent, an enzyme agent, a probiotic agent, a flavor agent, and a non-protein agent added to increase the efficacy Nitrogen compounds, silicates, buffers, colorants, extractants, oligosaccharides, and the like may be additionally included, and in addition, a feed mixture may be additionally included, but the present invention is not limited thereto.

The content of the oleifera extract in the feed additive composition may be appropriately selected according to the species, age, weight, and breeding conditions of the fed livestock, and is 0.01 to 95% by weight, preferably 0.1 to 80% by weight, based on the total weight of the feed additive composition. It may be a percentage by weight. The feed additive composition of the present invention may be prepared according to a method for producing a feed additive known in the art, and for example, it may be prepared by further performing molding into pellets or a cutting step in the form of granules. It is clear to those skilled in the art that the components, composition, manufacturing method, feeding method, etc. of the feed additive composition of the present invention can be appropriately selected from conventional techniques known in the art.

The present invention also provides an antiviral cleaning product containing an extract of Lonicera praeflorens as an active ingredient.

Since the extract of oleifera tree according to the present invention has the effect of inhibiting the proliferation of coronavirus, it is applied as a cleaning product of various formulations such as body soap, hand sanitizer, liquid shampoo, liquid soap, liquid rinse, body cleaner, cream soap, etc. can be In addition, its use can be applied to various fields. For example, it can be applied to hand sanitizer, mouthwash, whole body cleaner, dishware cleaner, residential environment cleaner, and the like. The various cleaning products of the present invention can be prepared in a liquid, creamy, or paste-like formulation by uniformly mixing and stirring the extract of oleracea in a conventional method in a conventional base composition, and compositions of other formulations are also common. A solid or spray-type product can also be manufactured according to a known method.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example 1. Preparation of leaf extract of olgobula tree

After drying the leaves purchased from the Korea Plant Extracts Bank, add 5 to 20 times distilled water based on the weight of the dried leaves, and bath for 0.5 to 3 hours at room temperature (25°C) to 100°C. Centrifugation was performed at 15,000 rpm for 10 to 30 minutes to remove insoluble substances, and the supernatant was freeze-dried to prepare a hot-water extract from oleracea. In addition, alcohol (e.g., ethanol or methanol) extracts from the leaves of the oleifera tree leaves are dried, and ethanol or methanol 5 to 25 times the weight of the dried hornbeam leaves is added, followed by 68 at room temperature to 60°C. After standing for ~77 hours, centrifugation was performed at 10,000 ~ 15,000 rpm for 15 ~ 30 minutes to remove insoluble precipitates, and the supernatant was dried and powdered.

In the present invention, ethanol 20 times the weight of the dried oleifera leaves is added, left at room temperature for 72 hours, and then subjected to ultrasonic treatment for 30 minutes to break up fine particles and centrifuged at 12,000 rpm for 20 minutes to recover only the supernatant and extract the ethanol extract was prepared.

Example 2. Analysis of antiviral activity of olgobulnae extract

2-1. Analysis of antiviral activity against PEDV of oleifera extract

In order to measure the virus proliferation inhibitory ability of the ethanol extract of oleracea prepared in Example 1 against PEDV (CV777 strain), Vero cells were cultured in a 96-well plate and each cell was at least 90% filled at the bottom of the well. At this time, remove the existing culture medium and treat each well with a new culture medium containing PEDV titrated with 100 TCID ₅₀ , and add ethanol extract of oleifera ethanol and antiviral compound AZT (azidothyymidine) or lamivudine to concentrations (0.1, 1, 10 and 100 μg/ml) were administered to the wells. Thereafter, the virus proliferation inhibitory ability was measured by SRB assay (Martin A, Martin C., Cytotechnology. 1993 11(1):49-58) of live cells after PEDV was infected with Vero cells for 48 hours. After adding 100 μl of 10% trichloroacetic acid (TCA) to each well, it was left at 4° C. for 1 hour and washed several times with distilled water. After drying at room temperature, 100 μl of a 0.4% (w/v) SRB (sulforhodamine B) solution dissolved in 1% (v/v) acetic acid was added and stained for 30 minutes. SRB staining solution not bound to cells was washed several times with 1% (v/v) acetic acid and then dried again. 100 μl of a 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the cell-bound dye, and then absorbance was measured at 560 nm. In each treatment group, the absorbance of the group that was not treated with the virus, the group treated only with the A. oleifera extract, the group treated only with the virus, and the group treated with the virus and the P. Antiviral activity was calculated. As a result, as shown in FIG. 1 , the antiviral activity of the olgopus extract was increased in a concentration-dependent manner, and the antiviral activity against PEDV at 100 μg/ml was 63.3%. In addition, it was confirmed that the antiviral activity of PEDV extracts of the oleifera extract was superior to that of AZT and lamivudine, which are antiviral compounds.

2-2. Analysis of antiviral activity against TGEV of oleifera extract

The virus proliferation inhibitory ability of the ethanol extract of oleracea obtained in Example 1 against TGEV (Purdue strain) was analyzed. The method was the same as in Example 2-1., and ST cells were used instead of Vero cells. As a result, as shown in FIG. 2 , the antiviral activity of the oleifera extract against TGEV increased in a concentration-dependent manner of that of the oleifera extract, and the antiviral activity against TGEV at 100 μg/ml was confirmed to be 58.33%. became

2-3. Analysis of antiviral activity against PRCV of oleifera extract

The virus proliferation inhibitory ability against PRCV (AR310 strain) of the ethanol extract obtained from Example 1 was analyzed. The method was the same as in Example 2-1., and ST cells were used instead of Vero cells. As a result, as shown in FIG. 3 , the antiviral activity against PRCV of the oleifera extract showed a tendency to increase in a concentration-dependent manner, and the antiviral activity against PRCV at 100 μg/ml was 70.28%. .

The homogeneity of the polymerase amino acid sequence between the PEDV, TGEV and PRCV coronaviruses is 50% or more, and if the amino acid sequence similarity is included, the similarity is very high, at 75% or more. Considering that the sequence similarity with -2 is 100%, the antiviral activity of the PEDV, TGEV and PRCV coronaviruses is applicable to other coronaviruses. It can be easily inferred that the composition comprising

Preparation Example 1. Capsules

10 mg of the extract obtained from Example 1 was mixed with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. The mixture was filled into No. 5 gelatin capsules using a suitable device.

Claims (7)

As an anti-coronavirus composition containing an ethanol extract of Lonicera praeflorens as an active ingredient,
The coronavirus is an anti-coronavirus composition, characterized in that it is a porcine epidemic diarrhea virus, a transmissible gastroenteritis coronavirus or a porcine respiratory coronavirus. delete delete Porcine epidemic diarrhea virus, Transmissible gastroenteritis coronavirus, or Porcine respiratory coronavirus infection prevention or treatment containing an ethanol extract of Lonicera praeflorens as an active ingredient pharmaceutical composition for use. Porcine epidemic diarrhea virus, Transmissible gastroenteritis coronavirus or Porcine respiratory coronavirus infection prevention or improvement containing ethanol extract as an active ingredient For health functional food composition. As an anti-coronavirus feed additive composition containing an ethanol extract of Lonicera praeflorens as an active ingredient,
The coronavirus is a swine epidemic diarrhea virus (Porcine epidemic diarrhea virus), infectious gastroenteritis coronavirus (Transmissible gastroenteritis coronavirus) or swine respiratory coronavirus (Porcine respiratory coronavirus) feed additive composition, characterized in that. As an antiviral cleaning product containing an ethanol extract of Lonicera praeflorens as an active ingredient,
The virus is a swine epidemic diarrhea virus (Porcine epidemic diarrhea virus), infectious gastroenteritis coronavirus (Transmissible gastroenteritis coronavirus) or porcine respiratory coronavirus (Porcine respiratory coronavirus) cleaning products, characterized in that.

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