WO2016159584A2 - Composition for innate immunity enhancement and antiviral activity containing extract of mori ramulus or mori radicis cortex as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for innate immunity enhancement and antiviral activity containing an extract of Mori Ramulus or Mori Radicis Cortex as an active ingredient, a health functional food for innate immunity enhancement and antiviral activity containing an extract of Mori Ramulus or Mori Radicis Cortex as an active ingredient, a feed composition for innate immunity enhancement and antiviral activity containing an extract of Mori Ramulus or Mori Radicis Cortex as an active ingredient, and a method for innate immunity enhancement and antiviral activity enhancement in an animal, the method comprising administering an extract of Mori Ramulus or Mori Radicis Cortex to an animal excluding humans, in need of innate immunity enhancement. The Mori Ramulus- or Mori Radicis Cortex-containing composition for innate immunity enhancement and antiviral activity according to the present invention exhibits antiviral activity and innate immunity enhancement efficacy, and thus can be applied for prevention and treatment of bacterial and viral infectious diseases, and can be widely used for a medicine or a health functional food for immunity enhancement and regulation in immunodeficient or immunosuppressed patients and for a feed for innate immunity enhancement and antiviral activity, for the purpose of anti-disease enhancement of animals. Furthermore, the composition for innate immunity enhancement and antiviral activity according to the present invention scarcely causes toxicity or side effects, and thus can be safely used even in the long-term use for prophylactic purposes.

Description

Congenital immunity enhancement and antiviral composition containing the extract of the upper limbs or lettuce extract as an active ingredient

The present invention relates to an innate immunity enhancing and antiviral composition containing the extract of the upper limbs or the lettuce extract as an active ingredient, more specifically, the extract of the upper limbs or lettuce extract, which can be used as a pharmaceutical, dietary supplement or feed composition as an active ingredient. It relates to innate immunity enhancement and antiviral composition.

Immunity can be divided into innate immunity, which has been born since birth, and acquired immunity, which is obtained by adapting to life after being acquired. Innate immunity, also known as 'natural immunity', is characterized by non-specific responses to antigens and no special memory function.

Recently, the field of innate immunity induced by interferon (interferon) in the innate defense immune system in vivo, because the activation of interferon-mediated immunity can be a fundamental preventive method against various infectious disease pathogens. Therefore, studies on the mechanism of interferon activation and the development of immunomodulators capable of inducing interferon are being actively conducted.

On the other hand, as one of the defense mechanisms of innate immunity following infection of the pathogen, immune factors such as cytokines are secreted, thereby causing an immune response and defense against the pathogen. Therefore, by inducing an innate immune response can be a method of prevention and treatment of various infectious disease pathogens, and research on the enhancement of innate immunity that can induce it.

Virus is a Latin word for toxic substance and is a group of infectious pathogenic particles that pass through bacterial filter paper (0.22㎛). Viruses may be classified as bacteriophages, plant viruses, or animal viruses according to host cell types, and may be classified as DNA viruses or RNA viruses according to nucleic acid types. Recently, various viral diseases such as swine flu, AI and foot-and-mouth disease caused social problems, and consequently, anxiety about effective measures for viral diseases has attracted great social attention.

Currently, the best way to prevent viral diseases is vaccination, but in the case of viral diseases, the effectiveness of vaccines due to the large number of virus serotypes (subtypes) is raised. Development and dissemination of inhibitors for the prevention of viruses that can supplement the problems of such vaccines is an important matter. For this purpose, the preventive agents that enhance the immunity of individual animals by stimulating the innate immune system, which is the initial defense system against viruses, are particularly important. Discovery and development can be an important method of drug development.

Representative antiviral agents that inhibit the growth of influenza viruses include amantadine and rimantadine, but these two antiviral agents are effective only against serotype A influenza virus and serotype B without M2 protein. Influenza virus was found to be ineffective. In addition, amantadine and rimantadine have been found to have the disadvantage that the emergence of a mutant virus that does not affect the ion channel function of the influenza virus M2 protein when used. To alleviate this drawback, zanamivir and oseltamivir have been developed as effective antiviral agents for all 16 serotype A influenza viruses and serotype B influenza viruses. However, zanamivir has the disadvantage of inhalation and intravenous administration, while oseltamivir is orally available, but has been pointed out as a disadvantage due to side effects such as recent reports of the emergence of resistant virus and vomiting and dizziness upon oral administration.

In addition, since the main control method of Vesicular stomatitis virus is impossible to treat the disease completely, prevention and blocking prevention and the eradication of susceptible livestock in the developing area is the best method, like foot-and-mouth disease.

In addition, vaccines against Newcastle disease virus are largely divided into live poison vaccine and dead poison vaccine. The most widely used representative Newcastle disease live vaccine vaccines, B1 and La Sota (including Clone), are known to induce vaccination response. Multivalent mixed poisoning oil vaccine, a globally used Newcastle disease poisoning vaccine, can prevent three or more diseases at the same time with a single vaccination. However, in case of laying hen farms, cases of newcastle disease are increasing due to reduced immunity.

There are also relatively small viruses among RNA viruses. They combine the word 'pico' which means small and 'RNA' to call piconaviruses, and the viruses belonging to them are collectively called the piconavirus family (Picona viridae). Enteroviruses belonging to the piconavirus family include about 70 serotypes that cause various clinical symptoms such as aseptic meningitis, hand and foot disease, herpes stenosis, dilated myocarditis, and acute hemorrhagic conjunctivitis. , Cossackie virus and Echo virus and other enteroviruses. Enteroviruses are about 20 to 30 nm in diameter and contain a single strand of RNA as a gene. Most of the infections in the digestive organs of vertebrates, as well as the respiratory tract and the central nervous system, but often do not show any obvious symptoms. Coxsackie virus (CXV) is a human enterovirus belonging to the family of piconaviruses. It is largely divided into types A and B (Pallansch MA and Roos RP, Fields Virology, 4th edi, pp723-775). , 2001).

In addition, recently, high-risk enteroviruses and mutant viruses (enterovirus type 71 (EV-71) and coxsackie A24 mutant strains) have been newly discovered and spread in various parts of the world. It is required.

Enteroviruses, including coxsackieviruses, infect the vertebrate digestive system, respiratory tract and central nervous system and cause various clinical symptoms. Therefore, it is urgently required to prepare measures at the national level, but the types and serotypes of viruses are very diverse. As such, no effective commercialized vaccines or therapeutics have been developed.

In addition, Herpes Simplex Virus (HSV) is a DNA virus belonging to the herpes virus, a relatively large virus having a size of about 175 nm, and is a widespread infectious agent in humans. Herpes simplex infection is an infection of HSV type 1 (hereinafter referred to as 'HSV-1') and HSV type 2 (hereinafter referred to as 'HSV-2'), which is an acute bullous disease involving the mucous membranes and skin. It is a common infectious disease in people. HSV-1 mainly forms around the mouth and eye, while HSV-2 forms blisters around the genitals. HSV infection is reported to be severe in patients with compromised immune function and also to cause cervical cancer (Melnick, JL, Adam, E. and Rawls, W., Concer, 34, 1355-1385, 1974). In addition, newborns and fetuses are not only able to produce HSV antibodies themselves, but because the antibodies of the mother are not passed on to the fetus, it is known that most infections have a fatal effect. Currently, about 500,000 cases of HSV-1-related eye disease occur in the United States alone, and more than 1,000 of them are undergoing ocular transplantation. About 95% of HSV-2 infections are known to be transmitted through sexual contact with a partner with active lesions. About 20-30% of US adults are infected with HSV-2, of which 20-50% relapse It has been reported to have genital herpes (Johnson RE et al., N. Engl. J. Med., 321, 7,1989).

HSV begins to replicate in the skin or mucosal epidermal cells after initial infection, migrates along the nerve tissue, lurks in the vertebral ganglion throughout life, and reactivates when the immune function is compromised. It is known to generate. Nucleoside derivative Acyclovir is known to be very effective for early infection as a therapeutic agent for HSV infection (Bryson, YJ et al., N. Engl. J. Med., 308, 916, 1983) In order to maintain the effectiveness of the drug, the drug must be continuously administered, and it is known that HSV relapses infectious diseases (Mindel A. et al., Lancet i: 926-928 (1988); Straus SE et al., N. Engl. J. Med., 310, 1545 (1984)). It has also been reported that mortality rates are high in patients with relapse after the initial infection of HSV (Nahmias, A. J. and Coleman, R. M., Immunobiology of Herpes Simplex Virus Infection CRC Press, Boca Raton, 92-102,1984).

Although vaccines have been developed to prevent such infections of HSV, live attenuated vaccines that use attenuated virus itself have been found to be directly related to the oncogenesis of HSV genomes. Use has been prohibited (Cappel, R., Sprecher, S., De Cuyper, F. and De Braekeleer, J., J. Med. Virol., 16, 137-145,1985).

Under these circumstances, many efforts have been made to overcome the shortcomings of existing antiviral agents in Korea and abroad, and among them, research on antiviral efficacy of herbal extracts and plant extracts is underway. However, since the current situation is inadequate, it is necessary to develop a composition using an herbal extract capable of overcoming the disadvantages of existing antiviral preparations and exhibiting excellent innate immunity promoting effect and antiviral activity with little toxicity and side effects. .

On the other hand, Mori Ramulus is a dried branch of mulberry plants. Mulberry plants include mulberry (Morus alba, Linne), drooping mulberry (Morus alba for. Peldulls DIPPEL), mountain mulberry (Morus bombycis, Koidzumi), tail mulberry (Morus bombycis var. maritima KOIDZ), Morus bombycis for Kase VYKEI, Mongolian mulberry, king mulberry, and stone mulberry (Morus tiliaefolia, Makino). It is distributed horizontally and grows near mountain villages or foothills. It is known that it has a skin whitening effect as it contains ingredients for branch action, antibacterial action, blood pressure lowering action, antidiabetic, and skin melanin cells.

In addition, Mori Radicis Cortex (Mori Radicis Cortex) is a medicinal herb made of mulberry or the root of the same plant as the medicinal herb to treat seawater, asthma caused by waste heat and has a diuretic effect. It is used for acute pyelonephritis and fragile edema. It has a blood pressure-lowering effect and is also used for nosebleeds and keratosis. It is also used for epidemic hepatitis. Pharmacological action is reported by Jinhae, diuresis, lowering blood pressure, sedation, analgesic, fever, jingyeong, antibacterial action. Morus alba Linne contains umberlin, umberlobogen, cyclomulberin and coumarin derivatives such as scopoline, umbeliferon, and triterpenoids such as amiline and betulinic acid. Antitussive, antibacterial, anti-inflammatory, antioxidant effects are known as excellent medicine. Morus alba L. and the cortex of the root of the same root rust plant were removed, and in winter, the roots were removed by digging the roots, and when it was fresh, peel off the cork layer and split it vertically and tap with a wood hammer to separate the neck and skin. Only drunk. The taste is sweet and the nature is cold. Epidermis has been used in oriental medicines to treat diabetes, diuretics, anti-emetics, diarrhea (Giaccia A. et al. Nat Rev Drug Disc 2; 803-811, 2003). The components include several compounds such as prenylated flavonoids, benzofuran, and phenolic compounds, which produce cytotoxicity, COX-1, COX-2, and nitric oxide (NO). Various biological activities have been reported, including inhibitory effects. The main bioactive components include umblliferone, mulberrochromene, cyclomulberrin and cyclomulberrochromene, and work with acetylcholine. It contains similar anti-inflammatory ingredients.

As an example of a technique that discloses the extract of the upper limb or baekryepi, the Korean Patent Publication No. 2003-0039521 discloses an antimicrobial composition comprising the extract of the upper limbs, and the upper limb extract and cheongung extract in Korean Patent No. 0635784 Disclosed is a cosmetic composition for skin whitening containing as a component, and disclosed in Korean Patent Publication No. 2012-0122429, a composition for treating pancreatic cancer comprising an extract of lettuce skin and a health functional food, and in Korean Patent No. 0902094 Disclosed is a pharmaceutical composition for preventing and treating cancer, which contains an extract of lettuce, its fractions and 2-arylbenzofuran compounds isolated from the fractions as an active ingredient.

However, there have been no reports on the effects of congenital immunity and antiviral activity using the extracts of the upper limbs or epithelium.

The present invention was derived by the above-mentioned demands, and the present invention completed the present invention by confirming that the upper limb or epithelium extract can inhibit the proliferation of various viruses by activating macrophages which are the main cells of innate immunity. In addition, the present invention has little toxicity and side effects, while ensuring long-term safety, the upper limb or lettuce extract can be effectively used in the pharmaceutical composition, health functional food or livestock feed composition for the prevention or treatment of infectious diseases caused by various viruses or bacteria It is an object of the present invention to provide a method for enhancing innate immunity and antiviral composition, enhancing innate immunity and antiviral activity of an animal.

In order to achieve the above object, the present invention provides a pharmaceutical composition for innate immunity enhancement and antiviral containing the upper limbs or lettuce extract as an active ingredient.

The present invention also provides a dietary supplement for innate immunity and antiviral, which contains the extract of the upper limbs or lettuce extract as an active ingredient.

In addition, the present invention provides a feed composition for innate immunity enhancement and antiviral containing the extract of the upper limbs or lettuce extract as an active ingredient.

The present invention also provides a method for enhancing innate immunity and antiviral activity of an animal, characterized by administering an extract of the upper limb or epithelium to an animal in need of enhancement of innate immunity except for humans.

According to the present invention can be effectively applied to the prevention and treatment of various viral and bacterial infectious diseases by using the extract of the upper limbs or epithelium, which exhibits excellent innate immunity promoting effect, it can contribute to the enhancement of immunity.

In addition, the composition for enhancing immunity and antiviral, including the upper limbs or extracts of the upper limbs according to the present invention can be used with confidence even for long-term use for prophylactic purposes because it rarely causes toxicity or side effects. Therefore, the composition according to the present invention can be applied for the prevention and treatment of infectious diseases caused by bacteria and viruses, pharmaceutical compositions or health functional foods for improving and regulating the immunity of patients with reduced or suppressed immunity, and of animals It can be widely used as feed composition for immuno-stimulation for the purpose of strengthening anti-disease.

In addition, according to the present invention can be effectively and safely promote the immunity of the animal by administering the upper limb or epithelium extract to the animal required for innate immunity enhancement.

1 is an antiviral activity analysis of influenza virus (PR8-GFP virus) of the extract of the upper limb according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; PR8-GFP is a virus infection group; IFN-β / PR8-GFP is a positive control group, including PR8-GFP virus infection and 1,000 Units / ml IFN-β treated group; Upper limb / PR8-GFP is a group of PR8-GFP virus infection and upper extremity extract treatment.

Figure 2 shows the results of the antiviral activity analysis of influenza virus (PR8-GFP virus) of the extract of the extract from the lettuce, according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; PR8-GFP is a virus infection group; IFN-β / PR8-GFP is a positive control group, including PR8-GFP virus infection and 1,000 Units / ml IFN-β treated group; Epithelium / PR8-GFP is a treatment group for PR8-GFP virus infection and epidermis extract.

Figure 3 shows the results of antiviral activity analysis of bullous stomatitis virus (VSV-GFP virus) of the extract of the upper limb according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; VSV-GFP is a virus infection group; IFN-β / VSV-GFP is a positive control group with VSV-GFP virus infection and 1,000 Units / ml IFN-β treated group; Upper limb / VSV-GFP is a VSV-GFP virus infection and upper limb extract treatment group.

Figure 4 shows the results of the antiviral activity analysis of the vesicular stomatitis virus (VSV-GFP virus) of the extract of the extract from the lettuce, according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; VSV-GFP is a virus infection group; IFN-β / VSV-GFP is a positive control group with VSV-GFP virus infection and 1,000 Units / ml IFN-β treated group; Epithelium / VSV-GFP is a VSV-GFP virus infection and epidermis extract treatment group.

5 is an antiviral activity analysis result for herpes simplex virus (HSV-GFP virus) of the extract of the upper limb according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; HSV-GFP is a virus infection group; IFN-β / HSV-GFP is a positive control group with HSV-GFP virus infection and 1,000 Units / ml IFN-β treated group; Upper limb / HSV-GFP is a HSV-GFP virus infection and upper limb extract treatment group.

Figure 6 is the result of antiviral activity analysis for herpes simplex virus (HSV-GFP virus) of the extract of the extract from the epidermis according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; HSV-GFP is a virus infection group; IFN-β / HSV-GFP is a positive control group with HSV-GFP virus infection and 1,000 Units / ml IFN-β treated group; Epidermis / HSV-GFP is a group treated with HSV-GFP virus infection and epidermis extract.

Figure 7 shows the results of the antiviral activity analysis of Newcastle disease virus (NDV-GFP virus) of the extract of the upper limb according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; NDV-GFP is a virus infection group; IFN-β / NDV-GFP is a positive control group with NDV-GFP virus infection and 1,000 Units / ml IFN-β treated group; Upper extremity / NDV-GFP is the NDV-GFP virus infection and upper extremity extract treated group.

Figure 8 shows the results of antiviral activity analysis of Newcastle disease virus (NDV-GFP virus) of the extract from the extract of lettuce, according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; NDV-GFP is a virus infection group; IFN-β / NDV-GFP is a positive control group with NDV-GFP virus infection and 1,000 Units / ml IFN-β treated group; Epithelium / NDV-GFP is a group of NDV-GFP virus infection and epidermis extract treatment.

9 is an antiviral activity analysis result for the enterovirus (EV-71 virus) of the extract of the upper limb according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; EV-71 is a viral infection group; IFN-β / EV-71 is a positive control group with EV-71 virus infection and 1,000 Units / ml IFN-β treated group; Upper extremity / EV-71 is the EV-71 virus infection and upper extremity extract treated group.

Figure 10 shows the results of the antiviral activity analysis of the enterovirus (EV-71 virus) of the extract of the extract from the lettuce, according to an embodiment of the present invention. Medium is a group of cells treated with nothing as a negative control; EV-71 is a viral infection group; IFN-β / EV-71 is a positive control group with EV-71 virus infection and 1,000 Units / ml IFN-β treated group; Epidermis / EV-71 is the EV-71 virus infection and epidermis extract treatment group.

Figure 11 shows the results of the pre-inflammatory cytokine induction analysis by extracting the upper limbs according to an embodiment of the present invention.

Figure 12 shows the results of the pre-inflammatory cytokine induction analysis by the extract from the lettuce extract according to an embodiment of the present invention.

13 is a result confirming the inhibition of infection of influenza viruses (H1N1 and H5N2) by the extract of the upper limb according to an embodiment of the present invention. H1N1 and H5N2 are influenza virus only (1.0 MOI) treated group, Medium is a negative control group treated with none of the MDCK cell group. ) Is processed simultaneously.

14 is a result confirming the suppression of infection of influenza viruses (H1N1 and H5N2) by the extracts of the epidermis extract according to an embodiment of the present invention. H1N1 and H5N2 were treated with influenza virus only (1.0 MOI), Medium was a negative control group treated with nothing but MDCK cell group. ) Is processed simultaneously.

The present invention relates to a pharmaceutical composition for innate immunity enhancement and antiviral containing the upper limbs or lettuce extract as an active ingredient.

The extract of the upper limbs or lettuce leaves is preferably extracted with one or more solvents selected from water and alcohols having 1 to 4 carbon atoms, more preferably with a solvent of water, methanol, ethanol or butanol, and even more preferably. Preferably, hot water is extracted using water as a solvent. The hot water extraction is 1) adding 5 to 30 times the distilled water based on the weight of the upper limbs; 2) extracting hot water at a temperature of 100-130 ° C. for 2-5 hours; And 3) filtering the hot water extract; but is not limited thereto.

The extract of the upper limbs or lettuce leaves may include any of the extract obtained by the extraction treatment, the dilution or concentrate of the extract, the dried product obtained by drying the extract, or the crude or purified product.

One example of a virus in which the upper limb or epithelium extract of the present invention has antiviral activity is orthomixoviridae, Rhabdoviridae, Paramixoviridae, herpesviridae and picona At least one virus selected from Picornaviridae, preferably influenza virus, Newcastle disease virus, Vesicular stomatitis virus, Cossackie virus, Enterovirus 71 (Enterovirus-71), simple Herpes Simplex Virus, Rhinovirus, Respiratory Syncytial Virus (RSV), Foot and Mouth Disease Virus, Colorado True Mite Virus, Leo Virus, Human Immunodeficiency Virus, Hepatitis B Virus, Hepatitis C Virus, Swine Fever Virus, Bovine Viral Diarrhea Virus (Bovine Viral Di) arrhea Virus, Porcine reproductive and respiratory syndrome virus, Porcine Ozeski's disease virus, Rotavirus, Parvovirus, Porcine epidemic diarrhea virus, and one more preferred virus is influenza. Virus, Newcastle disease virus, bullous stomatitis virus, herpes simplex virus, enterovirus type 71, rhinovirus, respiratory syncytial virus (RSV), but not limited thereto.

Upper limb or epithelium extract of the present invention exhibits strong immunity-inducing ability, increases the innate immunity of the individual and inhibits the infection and proliferation of the virus. Characterized in that can be used. When infecting pathogens, one of the defense mechanisms of innate immunity is to secrete immune factors.The secretion of these immune factors (TNF-α, IL-6 and IFN-β) protects the pathogens, thus inducing an appropriate level of cytokine response. Can be a prophylactic and treatment method for various infectious disease pathogens. In particular, it can enhance the immunity to the virus.

Pharmaceutical composition for innate immunity enhancement and antiviral of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. Pharmaceutically acceptable carriers, excipients or diluents which may be used in the present invention are not particularly limited so long as they do not impair the effects of the present invention, and include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, Sweeteners, fragrances, preservatives and the like. Representative examples of pharmaceutically acceptable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia rubber, alginate, calcium phosphate, calcium carbonate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable Ester, utopsol, macrogol, tween 61, cacao butter, lauridge, etc. are mentioned. Pharmaceutical composition for innate immunity enhancement and antiviral of the present invention may be in the form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and injections. The method of formulating the pharmaceutical composition may be carried out according to conventional methods known in the art, and is not particularly limited.

The pharmaceutical composition for congenital immunity enhancement and antiviral of the present invention may be administered orally or parenterally, and the dosage is depending on the age, sex, weight, condition, degree of disease, type of drug, route of administration and duration of administration. Although appropriately selected, generally about 5 to 500 mg / kg, preferably about 100 to 250 mg / kg may be administered once or three times a day.

It will be apparent to those skilled in the art that the method, dosage, route of administration, components, etc., of the innate immunity enhancing and antiviral pharmaceutical compositions of the present invention may be appropriately selected from conventional techniques known in the art.

The pharmaceutical composition for innate immunity and antiviral of the present invention can be used for the prevention and treatment of bacterial or viral infectious diseases. The pharmaceutical composition for innate immunity enhancement and antiviral of the present invention may be used in combination with a pharmaceutical composition including other pharmaceutically active ingredients in addition to the upper limbs or epithelium extract as an active ingredient, or other active ingredients.

In addition, the present invention relates to a dietary supplement for innate immunity and antiviral, including the upper limbs or lettuce extract. The dietary supplement for innate immunity and antiviral of the present invention may further comprise a food supplement acceptable food supplement. Food-acceptable food supplement additives which can be used in the present invention include sugars such as glucose, fructose, maltose, sucrose, dextrin, cyclodextrin and natural carbohydrates such as sugar alcohols such as xylitol, sorbitol, erythritol, taumate , Natural flavors such as stevia extract, synthetic flavors such as saccharin, aspartamic acid, colorants, pectic acid or salts thereof, alginic acid or salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohols, carbonating agents, and the like, but are not limited thereto. Health functional foods for innate immunity enhancement and antiviral of the present invention may be in the form selected from the group consisting of powders, granules, tablets, capsules, candy, chewing gum, jelly and beverages. The content of the upper limbs or lettuce extracts in the dietary supplement for innate immunity and antiviral may be appropriately selected in consideration of the form, flavor and taste of the food, for example 0.01 to 30 wt.% Based on the total weight of the dietary supplement. It may be in the range of%. It will be apparent to those skilled in the art that the form, composition, and manufacturing method of innate immunity enhancing and antiviral dietary supplements of the present invention may be appropriately selected from conventional techniques known in the art.

The present invention also relates to an innate immunity enhancing and antiviral feed composition comprising the extract of the upper limb or epithelium. The content of the upper limbs or lettuce extract in the feed composition for innate immunity and antiviral may be appropriately selected depending on the species, age, weight, and breeding conditions of the feed livestock, and 0.01 to 95% by weight relative to the total weight of the feed composition. Preferably it may be a ratio of 0.1 to 80% by weight. Feed composition for innate immunity and antiviral of the present invention may be prepared according to a feed production method known in the art, for example, after mixing various feed ingredients or blended feed with the extract of the upper limb or lettuce , Further processing steps, for example molding into pellets or cutting into granules and the like. It will be apparent to those skilled in the art that the components, compositions, methods of preparation, and methods of feeding innate immunity enhancing and antiviral feed compositions of the present invention may be appropriately selected from conventional techniques known in the art.

In addition, the present invention relates to a method for enhancing innate immunity and antiviral activity of an animal, characterized by administering an upper limb or an epithelium extract to an animal in need of enhancement of innate immunity except for humans. In the enhancement of innate immunity and antiviral activity of the animal of the present invention, the dosage, route of administration, timing of administration and the like of the upper limb or epithelium extract may be appropriately selected from conventional techniques known in the art. It is obvious.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Example 1 Preparation of Upper Limb or Morus Cortex Extract

The extracts of the upper and lower limbs were added 20 times of distilled water to the upper and lower limbs, respectively, based on the weight of each of the upper and lower limbs, and extracted by hot water at 115 ° C. for 180 minutes, first filtered at 0.45 μm, and then the second at 0.22 μm Filtration removed the precipitate. The pH was then adjusted to 7.0, 1 ml aliquoted into 1.5 ml Ep-tubes and stored at -20 ° C for use in all assays.

Example 2. Antiviral Activity Assay of Upper Limb or Epidermis Extract

Influenza virus (PR8), Vesicular stomatitis virus, Herpes simplex virus (HSV), Newcastle disease virus and Enterovirus 71 (Enterovirus-71; EV-71 Antiviral activity analysis of the upper limb or epithelium extract was performed.

(1) Antiviral Activity Assay

Influenza virus (PR8), bullous stomatitis virus (VSV), herpes simplex virus (HSV-GFP), and Newcastle disease virus (Raw 264.7 cells, 8 × 10 ⁵⁾ cell / well), and enterovirus 71 (Enterovirus-71; EV-71) was analyzed by infecting Hela cells.

After culturing the cells in a 12-well TC plate, DMEM to which 1% FBS was added was treated with 1 μg / ml of the upper limb or epithelium extract (pH adjustment) prepared in Example 1, respectively. The negative control was treated only with DMEM with 1% FBS, and the positive control was treated with mouse IFN-β (500 units / ml). After 12 hours of treatment of the upper or upper limb extract, PR8-GFP (MOI: 1.0), VSV-GFP (MOI: 1.0), HSV-GFP (MOI: 3.0), NDV-GFP (MOI: 3.0) and EV-71 ( MOI: 1.0) were inoculated respectively. After 2 hours of inoculation, the inoculum was removed, washed three times with PBS, and after 12 and 24 hours, the extent of virus infection was checked.

(2) Antiviral Activity Assay

1) Analysis result of influenza virus (PR8-GFP virus)

The results of antiviral activity assays for influenza virus of the upper limbs or epidermis extracts are shown in FIGS. 1 and 2. The results for the upper limb extracts are shown in FIG. 1, and the results for the extracts of the upper limbs are shown in FIG. 2.

Figure 1 (a) shows a green fluorescent protein (GFP) fluorescent image after 12 hours of infection, Figure 1 (b) shows a green fluorescent protein (GFP) fluorescent image after 24 hours of infection, Figure 1 (c) shows infection 12 Cell viability after hours and 24 hours is shown, and FIG. 1 (d) shows the number of viruses present in the cells. As a result of treatment with the upper limb extract, the infection rate of influenza virus was remarkably decreased, and the cell survival rate was also increased compared to the virus infection group (Fig. 1).

2 (a) shows a green fluorescent protein (GFP) fluorescence image 12 hours after infection, Figure 2 (b) shows a green fluorescent protein (GFP) fluorescence image 24 hours after infection. As a result of treatment with the extract of the epidermis, it was confirmed that the infection rate of the influenza virus was significantly decreased (FIG. 2).

2) Analysis result of bullous stomatitis virus (VSV-GFP virus)

Results of antiviral activity analysis for bullous stomatitis virus are shown in FIGS. 3 and 4. The results for the upper limb extracts are shown in FIG. 3, and the results for the extracts of the upper limbs are shown in FIG. 4.

Figure 3 (a) disclosed as a result of the upper limb extract shows a green fluorescent protein (GFP) fluorescent image after 12 hours of infection, Figure 3 (b) shows a green fluorescent protein (GFP) fluorescent image after 24 hours of infection, Figure 3 (c) shows the cell viability after 12 hours and 24 hours after infection, Figure 3 (d) shows the number of viruses present in the cell. As can be seen from Figure 3, as a result of treatment with the extract of the upper limbs, the infection rate of bullous stomatitis virus significantly decreased, the virus titer was also reduced, the cell survival rate was also increased compared to the virus infection group.

Figure 4 (a) disclosed as a result for the extract of the epithelium extract shows a green fluorescent protein (GFP) fluorescence image after 12 hours of infection, Figure 4 (b) shows a green fluorescent protein (GFP) fluorescence image after 24 hours of infection . As can be seen from Figure 4, as a result of treatment with the extract of the epidermis, it was confirmed that the infection rate of the bullous stomatitis virus significantly dropped.

3) Analysis result of herpes simplex virus (HSV-GFP virus)

Results of antiviral activity analysis for herpes simplex virus are shown in FIGS. 5 and 6. The results for the upper limb extracts are disclosed in FIG. 5, and the results for the extracts of the upper limbs are disclosed in FIG. 6. Figure 5 (a) disclosed as a result of the upper limb extract shows a GFP fluorescence image 12 hours after infection, Figure 5 (b) shows a GFP fluorescence image after 24 hours of infection, Figure 5 (c) shows the infection 12 hours and Cell viability after 24 hours is shown, and FIG. 5 (d) shows the number of viruses present in the cells. As can be seen from Figure 5, as a result of treatment with the extract of the upper limbs, the infection rate of herpes simplex virus significantly decreased, the cell survival rate also increased compared to the virus infected group.

Figure 6 (a) disclosed as a result for the extract of the epithelium extract shows a GFP fluorescence image 12 hours after infection, Figure 6 (b) shows a GFP fluorescence image 24 hours after infection. As can be seen from Figure 6, as a result of treatment with extracts of the epidermis extract, it was confirmed that the infection rate of herpes simplex virus significantly decreased.

4 ) Analysis result of NDV-GFP virus

Results of antiviral activity assays for Newcastle Disease Virus are shown in FIGS. 7 and 8. The results for the upper limb extracts are shown in FIG. 7, and the results for the extracts of the upper limbs are shown in FIG. 8.

Figure 7 (a) shows the results of the upper limb extract GFP fluorescence image 24 hours after infection, Figure 7 (b) shows the relative amount of GFP fluorescence. As shown in FIG. 8, the results of the extract of the epidermis extract showed that the Newcastle disease virus was significantly reduced as a result of the treatment of the upper limb extract on the cells infected with the Newcastle disease virus.

5 ) Analysis result of enterovirus (EV-71 virus) type 71

Results of antiviral activity analysis for enterovirus 71 are shown in FIGS. 9 and 10. The results for the upper limb extracts are shown in FIG. 9, and the results for the extracts of the upper limbs are shown in FIG. 10.

9 (a) shows the optical image after 12 hours of infection, and FIG. 9 (b) shows an optical image after 24 hours of infection.

As for the results of the extract of the epithelium extract, as shown in FIG. 10, as a result of treating the upper limb extract on the cells infected with enterovirus 71, it was confirmed that enterovirus 71 was significantly reduced.

Example 3 Analysis of Pro-inflammatory Cytokine Induction of Upper Limb or Epidermis Extract Using Mouse Macrophage Cell Line

In order to confirm the immune factor induction effect of the upper limbs or epithelium extract, a pre-inflammatory cytokine induction assay was performed.

(1) Inflammatory cytokine induction assay

Raw 264.7, a mouse macrophage line, was grown and used for analysis. Cells were cultured in 6-well TC plates and then treated by adding the prepared upper limb extract (pH adjusted) to DMEM with 1% FBS. The negative control group treated only DMEM with 1% FBS, and the positive control group was a cytokine secreted in response to foreign substances such as viruses and cancer cells. Interferon, a substance that induces an anti-cancer and antiviral action and induces an immune response -β (IFN-β) was treated at 1000 Units / ml. Samples were treated with 1 μg / ml of the upper limb or epithelium extract, and the production amount (pg / ml) of TNF-α, IL-6 and INF-β was measured by ELISA on cells after 12 hours and 24 hours.

(2) Results of whole inflammatory cytokine induction assay

The results of the analysis of whole inflammatory cytokine induction by the upper limb or epithelium extract are shown in FIGS. 11 and 12. Tumor necrosis factor-α (TNF-α) is mainly secreted by activated macrophages, the most important role is the regulation of immune cells. It is also known to have the ability to inhibit viral replication. Interleukin 6 (IL-6) is an important cytokine that activates B-cells to increase antibody production and promote antigen-specific immune responses Interferon (IFN) is a cell regulator that functions very well. For example, it protects cells from viruses, inhibits cell division in tissue culture or bone marrow, regulates T cell function, induces the functioning of natural immune cells (NK cells), increases phagocytosis, and special cancer cells. It is known to suppress cleavage of.

As shown in FIG. 11, it was confirmed that TNF-α, IL-6, and IFN-β, which are proinflammatory cytokines, were strongly induced by the extract of the upper limbs in Raw 264.7 cells. In 264.7 cells, it was confirmed that the inflammatory cytokines IL-6 and IFN-β were strongly induced by the extract from the epidermis.

Example 4 Analysis of Inhibition of H1N1 and H5N2 Virus Infection by Upper Limb or Epidermis Extract

(1) Analysis method

MDCK cell line was treated with 1 μg / ml of the upper limb or epithelium extract and 1.0 multiplicity of infection virus at the same time. After 24 hours, 10 μl of Ez-Cytox reagent was treated and incubated for 12 hours to measure cytotoxicity. Absorbance was measured at 450 nm.

(2) Analysis result

Analysis of cell viability caused by H1N1 and H5N2 virus infection by upper limb or epithelial extract showed that the cell viability was reduced to about 20% by H1N1 and H5N2 virus infection, and 1 μg / ml of upper limb or epithelial extract and 1 MOI. In the group treated with the virus at the same time, the cell survival rate was 80-90%, it was confirmed that prevents the decrease in cell survival rate by virus infection (Figs. 13 and 14).

Preparation Example 1 Injection

Upper extremity or lettuce extract prepared in Example 1: 100 mg

Sodium metabisulfite: 3.0 mg

Methylparaben: 0.8 mg

Propylparaben: 0.1 mg

Sterile Distilled Water for Injection: Appropriate

The ingredients were mixed and prepared in a conventional manner to a final volume of 2 ml, filled in ampoules and sterilized to prepare an injection.

Preparation Example 2 Tablet

Upper limb or lettuce extract prepared in Example 1: 200 mg

Potato starch: 100 mg

Lactose: 100 mg

Colloidal silicic acid: 16 mg

Magnesium Stearate: Appropriate

The above ingredients were mixed, compressed into tablets and prepared according to a conventional tablet preparation method.

Preparation Example 3 Capsule

Upper extremity or lettuce extract prepared in Example 1: 100 mg

Lactose: 50 mg

Starch: 50mg

Talc: 2 mg

Magnesium Stearate: Appropriate

The capsules were prepared by mixing the ingredients and filling the gelatin capsules according to a conventional capsule production method.

Preparation Example 4 Pill

Upper extremity or lettuce extract prepared in Example 1: 120 mg

Corn starch: 100 mg

Sterilized Distilled Water: Appropriate

The above ingredients were mixed and refilled into spheres having the appropriate size according to a conventional pill preparation method to prepare pills.

Preparation Example 5 Health Functional Foods

1) health drink

Oligosaccharide (2%), liquid fructose (0.5%), sugar (2%), salt (0.5%), water (75%) and other beverage ingredients such as the extract of the upper limbs or lettuce extract prepared in Example 1 The beverage was prepared by sterilization.

2) functional food

Upper and lower limb extract prepared in Example 1 was properly mixed with various vitamins and mineral-containing functional food to prepare a functional food containing the upper limb or lettuce extract.

Preparation Example 6 Feed Composition

An appropriate amount of the upper limb or lettuce extract prepared in Example 1 was mixed with the mixed animal feed, to prepare a feed composition, and then pelletized and granulated.

Claims (13)

1. Congenital immunity enhancement and antiviral pharmaceutical composition, characterized in that it contains the extract of the upper limbs or lettuce extract as an active ingredient.
2. The method according to claim 1, wherein the upper limbs or baekryepi extract is water, a pharmaceutical composition for enhancing immunity and antiviral, characterized in that the extraction with one or more solvents selected from alcohols having 1 to 4 carbon atoms.
3. The method of claim 1, wherein the virus is at least one virus selected from Orthomixoviridae, Rhabdoviridae, Paramixoviridae and Paracornoviridae and Picornaviridae. Pharmaceutical composition for enhancing innate immunity and antiviral.
4. According to claim 3, Orthomixoviridae is Influenza virus (Influenza virus), Rhabdoviridae (Rhabdoviridae) is Vesicular stomatitis virus (Vesicular stomatitis virus), Paramixoviridae (Paramixoviridae) is Newcastle disease virus (Newcastle disease virus), Piconaviridae (Picornaviridae) is enterovirus 71 (Entero virus 71), characterized in that the innate immunity enhancement and antiviral pharmaceutical composition.
5. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
6. The method of claim 1, wherein the pharmaceutical composition is in tablets, pills, powders, granules, capsules, suspension emulsions, syrups, aerosols, external preparations, suppositories, and injections, characterized in that it has a formulation selected from the group consisting of Pharmaceutical composition for antiviral.
7. Congenital immunity enhancement and anti-viral health functional foods containing the extracts of the upper limbs or lettuce extract as an active ingredient.
8. 8. The dietary supplement for innate immunity and antiviral of claim 7, wherein the dietary supplement further comprises a food supplement acceptable food supplement.
9. 8. The dietary supplement for innate immunity and antiviral of claim 7, wherein the dietary supplement has a formulation selected from the group consisting of powders, granules, tablets, capsules, candy, chewing gum, jelly and beverages.
10. The method of claim 7, wherein the virus is at least one virus selected from Orthomixoviridae, Rhabdoviridae, Paramixoviridae (Paramixoviridae) and Piconaviridae (Picornaviridae) Congenital immunity and antiviral health functional foods.
11. The method of claim 10, wherein the Orthomixoviridae is an Influenza virus, the Rhabdoviridae is a Vesicular stomatitis virus, and the Paramixoviridae is 11. Newcastle disease virus (Picornaviridae) and Entero virus 71 (Picornaviridae) is a health functional food for innate immunity and antiviral, characterized in that the Entero virus 71.
12. Congenital immunity enhancement and antiviral feed composition containing the extract of the upper limbs or lettuce extract as an active ingredient.
13. A method of enhancing innate immunity and antiviral activity of an animal, characterized by administering the extract of the upper limb or epithelium to an animal in need of enhancement of innate immunity except for humans.

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