KR102210919B1 - A composition for the improving, preventing and treating of influenza virus infection comprising polysaccharide fraction isolated from barley leaf - Google Patents

Abstract

The present invention relates to a composition for improving, preventing or treating influenza virus infectious diseases, and contains a polysaccharide fraction derived from barley leaves as an active ingredient, so that the effect of preventing or treating influenza virus infectious diseases is excellent, preventing influenza virus infection diseases Or it can be usefully used as a pharmaceutical composition and food for treatment.

Description

A composition for the improving, preventing and treating of influenza virus infection comprising polysaccharide fraction isolated from barley leaf}, containing a polysaccharide fraction derived from barley leaf as an active ingredient.

The present invention relates to a composition having an excellent effect of improving, preventing or treating influenza virus infection diseases by containing a polysaccharide fraction derived from barley leaf as an active ingredient.

Influenza virus is one of the most important pathogens in public health as a cause of flu, an acute respiratory infection.

The seasonal influenza virus, consisting of the H1N1 and H3N2 subtypes of influenza A virus and the Victoria and Yamagata lineage B type influenza virus, is a winter epidemic every year and causes great damage to people. According to the World Health Organization (WHO), about 3 million to 5 million people worldwide each year become infected with the seasonal influenza virus and suffer serious illnesses, of which 250,000 to 500,000 people die.

The pandemic of the influenza virus causes even more damage. About 50 million people died during the 1918 influenza virus pandemic, and about 1 million people died during the 1957 and 1968 influenza virus pandemics. Humanity experienced serious human damage, economic damage, and social turmoil even during the pandemic caused by the new influenza virus in 2009.

The avian influenza virus is also considered a serious threat. Even today, cases of continuous human infection of the avian influenza virus have been reported. In particular, the highly pathogenic H5N1 avian influenza virus is analyzed to have a high mortality rate of about 59%, and the H7N9 avian influenza virus is analyzed to have a high mortality rate of about 32%.

The most effective method for preventing such influenza virus is known as vaccination, but there is a limit to controlling the damage caused by influenza virus only with vaccine. Despite being vaccinated against the seasonal influenza virus, 250,000 to 500,000 deaths are issued annually worldwide.

In the case of influenza vaccines, due to the antigenic mutation characteristics of the influenza virus, it is produced according to a production system that predicts a virus that will pandemic in the winter of that year at the beginning of the year and then produces a vaccine against it. Therefore, a situation may arise that the produced vaccine does not match the antigen of the influenza virus that is pandemic that winter, and in this case, it may not be able to effectively protect humans from influenza virus infection. In addition, if the swine flu virus suddenly outbreaks at an unexpected time, it is difficult to respond quickly with a vaccine.

The use of antiviral agents was able to overcome the limitations of these vaccines. In fact, in the case of the 2009 H1N1 pandemic, oseltamivir (NA inhibitor, NAI), an antiviral agent (NA inhibitor, NAI) that inhibits neuraminidase (NA), one of the surface glycoproteins of the influenza virus, before a sufficient dose of vaccine is prepared. Tamiflu®) has been widely prescribed as an effective means of controlling the swine flu virus. According to a recent study, it was analyzed that the prescription of oseltamivir at the time of 2009 effectively protected people from the swine flu virus.

Korea is also highly required to develop new antiviral drugs with domestic technology, and new antiviral candidates in Korean traditional drugs or foods to accumulate domestic unique technology and reduce dependence on foreign countries for antiviral drug development. There is an urgent need for research to search for and discover.

Korean Registered Patent No.1338319 Korean Patent Registration No. 1077920

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of influenza virus infection diseases containing a polysaccharide fraction derived from barley leaves as an active ingredient.

In addition, another object of the present invention is to provide a food composition for preventing or improving influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaves as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for animals other than humans for the prevention or treatment of influenza virus infection diseases containing a polysaccharide fraction derived from persimmon leaves as an active ingredient.

In addition, another object of the present invention is to provide a feed composition for preventing or improving influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaves as an active ingredient.

In addition, another object of the present invention is to provide a feed additive composition for preventing or improving influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaves as an active ingredient.

In order to achieve the above object, the pharmaceutical composition for preventing or treating influenza virus infection diseases of the present invention may contain a polysaccharide fraction derived from barley leaves as an active ingredient.

The polysaccharide fraction derived from barley leaves is 65 to 85% by weight of neutral sugar, 5 to 30% by weight of uronic acid, and 2-keto-3-deoxy-D-manno-octulosonic (KDO) relative to the total polysaccharide fraction. acid) may contain 0.5 to 10% by weight of a KDO-like substance.

The polysaccharide fraction derived from barley leaves may further contain 0.5 to 20% by weight of protein.

The uronic acid is composed of galacturonic acid and glucuronic acid; The neutral polysaccharide may include rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose.

The pharmaceutical composition may inhibit neuraminidase activity, preferably H1N1, H3N2, H4N2, H5N2, H6N2 and H7N2 neuraminidase activity.

The disease caused by the influenza virus infection may be at least one selected from the group consisting of flu, cold, sore throat, bronchitis, and pneumonia.

The flu may be at least one selected from the group consisting of bird flu, swine flu and swine flu.

The polysaccharide fraction comprises the steps of (a) treating barley leaf powder with pectin hydrolase; And (b) recovering the fraction by precipitating the enzyme-treated barley leaf powder with C1 to C4 alcohol.

The manufacturing method may further include (c) recovering a fraction having a molecular weight of 30 kDa or more from the polysaccharide fraction of step (b).

In addition, the food composition for preventing or improving influenza virus infectious diseases of the present invention for achieving the other object described above may contain a polysaccharide fraction derived from barley leaves as an active ingredient.

In addition, the pharmaceutical composition for animals other than humans for the prevention or treatment of influenza virus infection diseases of the present invention for achieving the above-described another object may contain a polysaccharide fraction derived from barley leaves as an active ingredient.

The animal influenza virus infectious diseases include mutant infectious bird flu, Avian paramyxoovirus infection caused by newcastle disease virus, Canine distemper infection, chicken infectious broncheitis virus. Turkey coronavirus infection caused by avian pneumovirus, avian pneumovirus infection caused by avian pneumovirus, pig genital and respiratory syndrome virus infection caused by porcine reproductive and respiratory syndrome virus, Cock Foot-and-mouth disease due to coxsakie virus, Avian leukosis due to reticuloendotheliosis virus, Chicken infectious laryngeal tracheitis due to aujeszky's disease virus infectious laryngotrachitis), respiratory infections caused by adenovirus, and animal adenovirus infections that cause egg drop syndrome.

In addition, the feed composition for preventing or improving influenza virus infection diseases of the present invention for achieving the above other object may contain a polysaccharide fraction derived from barley leaves as an active ingredient.

In addition, the feed additive composition for preventing or improving influenza virus infection disease of the present invention for achieving the other object described above may contain a polysaccharide fraction derived from barley leaves as an active ingredient.

The composition for improving, preventing, or treating influenza virus infection disease containing a polysaccharide fraction derived from barley leaf of the present invention has excellent effect of improving, preventing or treating influenza virus infection by inhibiting neuraminidase enzyme activity of influenza virus. , It can be usefully utilized as a pharmaceutical composition, food, feed composition, feed additive composition, etc. for the improvement, prevention or treatment of influenza virus infection.

1 is a graph showing the results of an enzyme-treated crude polysaccharide fraction (BLE-0) prepared according to Example 1 for inhibition of influenza virus (H1N1) neuraminidase activity.
Figure 2 is a neurami for various subtypes of influenza viruses (H3N2, H4N2, H5N2, H6N2, H7N2) derived from animals and humans of the enzyme-treated crude polysaccharide fraction (BLE-0) prepared according to Example 1 of the present invention. It is a diagram showing the inhibition of nidase activity.
3 is a graph showing the titer of the virus remaining in the tissues of mice for each group.

The present invention relates to a composition having an excellent effect of improving, preventing or treating influenza virus infection diseases by containing a polysaccharide fraction derived from barley leaf as an active ingredient.

Hereinafter, the present invention will be described in detail.

The composition capable of improving, preventing or treating infection against influenza virus of the present invention contains a polysaccharide fraction derived from barley leaves as an active ingredient.

The polysaccharide fraction derived from barley leaf of the present invention contains 65 to 85% by weight of neutral sugar, 5 to 30% by weight of uronic acid, and 2-keto-3-deoxy-D (KDO) relative to the total polysaccharide fraction. -manno-octulosonic acid) contains 0.5 to 10% by weight of a KDO-like substance. In addition, it may further contain 0.5 to 20% by weight of protein.

The uronic acid is composed of galacturonic acid and glucuronic acid; The neutral polysaccharide may include rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose, but is not limited thereto.

Preferably, the neutral polysaccharide is 30 to 50 mole% of xylose, 10 to 30 mole% of arabinose, 1 to 20 mole% of galactose, 1 to 10 mole% of glucose relative to the total neutral polysaccharide constituting the barley leaf-derived polysaccharide fraction. , Rhamnose 1 to 10 mole%, mannose 0.4 to 10 mole%, and fucose 0.1 to 7 mole% of neutral saccharides.

The polysaccharide fraction derived from barley leaf of the present invention comprises the steps of: (a) treating barley leaf powder with pectin hydrolase; And (b) recovering the fraction by precipitating the enzyme-treated barley leaf powder with C1 to C4 alcohol.

First, in step (a), barley leaf powder is treated with pectin hydrolase. The pectin hydrolase may be added in an amount of 1 to 4% by weight, preferably 1 to 3% by weight, and more preferably 1.5 to 2.5% by weight based on the weight of the barley leaf powder.

In the present invention, barley leaves may be undried or dried, and pulverized or powdered may be used. Preferably, the barley leaf powder treated with the enzyme may be used by suspending it in distilled water about 10 to 30 times (w/v).

The hydrolytic enzyme treatment is preferably performed for 1 to 4 days, more preferably 2 to 3 days.

The step (a) may further perform a process of inactivating the enzyme by heating the remaining pectin hydrolase at 90 to 100° C. for 10 to 60 minutes after the enzyme treatment. Due to the heating, the elution of the soluble polysaccharide component is increased, and the polysaccharide extract obtained by centrifugation and purity is improved by denaturing and precipitating a polymer protein included as some impurities.

Since the barley leaf extract obtained through the step (a) undergoes a process of hydrolytic enzyme treatment, the ratio of neutral polysaccharide is remarkably higher than that of the extract obtained by hot water extraction of barley leaf as it is. That is, the barley leaf extract extracted according to the above method contains a large amount of a polysaccharide fraction.

In addition, after step (a), a step of removing the residue from the hydrolyzed barley leaf extract may be additionally performed. The method of removing the residue may be performed by a method of removing solids from a known mixture such as centrifugation and filtration, preferably centrifugation.

Next, in step (b), an organic solvent is added to the enzyme-treated barley leaf hydrolyzate to obtain a precipitated polysaccharide. The organic solvent may be a C1 to C4 alcohol. Alcohol precipitation may be performed by a known ethanol precipitation method, and ethanol used for ethanol precipitation is preferably ethanol having a concentration of 70% to 95% (v/v) by mixing with water.

The manufacturing method may further include (c) recovering a fraction having a molecular weight of 30 kDa or more using ultrafiltration or gel filtration chromatography after step (b).

The method of obtaining the fraction is not particularly limited as long as it is a method of purifying on the basis of molecular weight, but is preferably ultrafiltration or gel filtration chromatography, more preferably gel filtration chromatography. In addition, the form of the final polysaccharide fraction may be an extract, a concentrate, or a powder.

A step of purifying the polysaccharide by removing low molecular weight substances and impurities by adding 50 to 100% alcohol having 1 to 4 carbon atoms to the final polysaccharide fraction may be further performed.

As a rescue chuck, the present invention is a crude polysaccharide fraction derived from barley leaves having a molecular weight of 30 kDa or more using the same method as described above by treating a barley leaf-derived fraction obtained by treatment with the pectinase and precipitating alcohol having 1 to 4 carbon atoms ( BLE-0) was obtained.

As a result of analyzing the constituent sugar of the obtained barley leaf-derived crude polysaccharide fraction (BLE-0) by gas chromatography, compared to the total polysaccharide, 74.77 wt% neutral polysaccharide, 19.37 wt% uronic acid, 3.66 wt% protein, 2.19 KDO-like substances % By weight; As a result of analyzing the components of the neutral polysaccharide, relative to the total neutral polysaccharide, rhamnose 3.84 mole%, fucose 0.6 mole%, arabinose 16.56 mole%, xylose 37.27 mole%, mannose 1.15 mole%, galactose 11.10 mole%, glucose It was confirmed that it was 4.25 mole% (refer to [Table 2]).

Compositions containing the barley leaf-derived polysaccharide fraction as an active ingredient include pharmaceutical compositions capable of preventing or treating influenza virus infection diseases, food compositions capable of preventing or improving influenza virus infection diseases, prevention or treatment of influenza virus infection diseases It may be a pharmaceutical composition, feed composition or feed additive composition for animals other than humans. The composition includes H1N1, H3N2, or H5N1 in the serotype of the influenza virus having neuraminidase inhibitory activity, preferably H1N1, but is not limited thereto.

In addition, the disease caused by the influenza virus infection may include at least one selected from the group consisting of flu, cold, sore throat, bronchitis, and pneumonia, but is not limited thereto. Specifically, the flu may include at least one selected from the group consisting of bird flu, swine flu, and swine flu, but is not limited thereto.

The term “fraction” used in referring to barley leaves in the present specification includes not only the fraction obtained by treatment with an extraction solvent, but also the processed product of the polysaccharide fraction derived from barley leaves. For example, the polysaccharide fraction derived from barley leaves may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the barley leaf-derived polysaccharide fraction of the present invention has a broad meaning of including a barley leaf polysaccharide processed product, for example, a barley leaf polysaccharide powder formulated to be administered to an animal. Although the experiment was conducted with the polysaccharide fraction derived from barley leaf in the present invention, it will be expected by those skilled in the art that the desired effect can be achieved even in the same form as the processed barley leaf polysaccharide.

Meanwhile, in the present specification, the term “contained as an active ingredient” means containing an amount sufficient to achieve the efficacy or activity of the polysaccharide fraction derived from barley leaf. As an example, the polysaccharide fraction derived from barley leaves is used in a concentration of 1 to 30 mg/kg, preferably 3 to 20 mg/kg. Since the barley leaf-derived polysaccharide fraction is a natural product and does not have side effects on the human body even when administered in an excessive amount, the upper limit of the quantity of the barley leaf-derived polysaccharide fraction included in the composition of the present invention can be selected and carried out within an appropriate range by a person skilled in the art.

The pharmaceutical composition or veterinary pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes excipients, disintegrants, sweeteners, binders, coating agents, Expanding agents, lubricants, lubricants or flavoring agents may be used.

The pharmaceutical composition or the pharmaceutical composition for animals may be preferably formulated into a pharmaceutical composition including at least one pharmaceutically acceptable carrier in addition to the above-described active ingredient for administration.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition or veterinary pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably Is oral administration.

Suitable dosages of the pharmaceutical composition or veterinary pharmaceutical composition of the present invention include formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response of the patient (or animal to be treated). It varies depending on factors such as sensitivity, and usually a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition or veterinary pharmaceutical composition of the present invention may be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a food composition for preventing or improving influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaves as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gum, ice cream, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include not only a barley leaf-derived polysaccharide fraction as an active ingredient, but also an ingredient commonly added during food production, for example, protein, carbohydrate, fat, nutrients, seasoning and flavoring agents. Include. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts, etc., in addition to the polysaccharide fraction derived from barley leaf of the present invention may be additionally included. I can.

The present invention provides a health functional food containing a food composition containing the barley leaf-derived polysaccharide fraction as an active ingredient. Health functional foods are foods prepared by adding a polysaccharide fraction derived from barley leaf to food materials such as beverages, teas, spices, gums, confectionery, or encapsulated, powdered, or suspended. It means to bring it, but unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of polysaccharide fraction derived from barley leaf in such health functional foods cannot be uniformly regulated depending on the type of health functional food to be targeted, but can be added within the range that does not damage the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In addition, the present invention provides a feed composition for preventing or improving influenza virus infection disease containing a polysaccharide fraction derived from barley leaf as an active ingredient.

In addition, the present invention provides a feed additive composition for preventing or improving influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaves as an active ingredient.

In addition, the present invention provides a use of a polysaccharide fraction derived from barley leaves for the manufacture of medicines or foods for improving, preventing or treating influenza virus infectious diseases. As described above, the barley leaf-derived polysaccharide fraction may be used for improvement, prevention or treatment of influenza virus infectious diseases.

In addition, the present invention provides a method for improving, preventing or treating influenza virus infection diseases comprising administering an effective amount of a polysaccharide fraction derived from barley leaves to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experiment, and preferably refers to a human.

In addition, the “animals other than humans” include mammals such as pigs, cows and goats: fish such as carp and goldfish: and poultry such as pheasants, chickens, ducks, and turkeys.

The present invention provides a feed composition for preventing or improving influenza virus infection diseases comprising a polysaccharide fraction derived from barley leaf as an active ingredient.

In addition to the polysaccharide fraction derived from barley leaf, the'feed composition' can use food ingredients and food additives described in the Food Additives Code, which can be used as foods described in the standards and standards of food ('Food Code'), as an active ingredient. Even if it is not a usable food raw material or food additive, raw materials that fall within the range of sweet feeds in Appendix 1 of'Standards and Specifications for Feed, etc.' and raw materials that fall within the range of supplementary feeds in Appendix 2 can be used.

The'feed composition' may be an extractant among auxiliary feeds according to'standards and standards for feed, etc.', and may be a compounded feed including the auxiliary feed.

In the case of preparing a feed composition using the barley leaf-derived polysaccharide fraction as an active ingredient, the barley leaf-derived polysaccharide fraction does not need to be particularly limited as long as it has an effect of preventing or improving influenza virus infection disease, but, for example, 0.1 to 99% by weight , 0.5 to 95% by weight, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, may be included in 5 to 50% by weight.

The polysaccharide fraction derived from barley leaves, which is an active ingredient in the feed composition, varies depending on the condition, weight, and the presence or absence of disease and the duration of the ingested animal, but may be appropriately selected by a person skilled in the art. For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, most preferably 50 to 500 mg based on the daily dosage. In addition, the number of administrations does not need to be particularly limited, but can be adjusted by a person skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be less than the above range.

The term “effective amount” as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human, which is considered by a researcher, veterinarian, doctor or other clinician, Includes an amount that induces relief of symptoms of the disease or disorder. The effective amount and the number of administrations for the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by a person skilled in the art, and the type of disease, severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health condition of the patient. , Sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and drugs used simultaneously. In the prevention, treatment, or improvement method of the present invention, in the case of adults, when the polysaccharide fraction derived from barley leaves is administered once to several times a day, it is preferable to administer it in a dose of 0.001 g/kg to 10 g/kg.

In the treatment method of the present invention, the composition comprising a polysaccharide fraction derived from barley leaves as an active ingredient is conventionally used through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered in a phosphorus manner.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention, but the following examples are only illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It is natural that such modifications and modifications fall within the appended claims.

Example 1. Barley leaf-derived crude polysaccharide fraction (BLE-0)

Barley grass was produced in Yeonggwang, Jeollanam-do, and was purchased and used by Saemomwon Co., Ltd.

Powdered barley is suspended in distilled water 20 to 30 times (w/v) (pH 4), and pectinase (Rapidase C80MAX, non-biochem) is added in an amount of 1 to 3% per raw material. Enzymatic treatment was performed for 3 days. Thereafter, the enzyme-treated reaction solution was heat-treated at 90 to 100°C for 30 to 60 minutes to inactivate the remaining pectinase.

After the enzyme treatment was completed, the sample was centrifuged at 4° C., 6,500×g for 15 minutes to remove the residue, and 4 times the volume (v/v) of ethanol was added thereto, and polysaccharide was precipitated while standing for 4 hours. The precipitated polysaccharide was recovered, and then the precipitated polysaccharide was dissolved in a small amount of distilled water, and then low molecular weight substances were removed using a dialysis membrane (MWCO 6000-8000), and an enzyme-treated polysaccharide fraction (BLE-0) was obtained.

Comparative Example 1. Crude polysaccharide fraction derived from barley leaves (BLW-0)_non-enzyme treatment (hot water extraction)

Powdered barley was suspended in distilled water 20 to 30 times (w/v) and heated at 100° C. for 3 hours. The hot water extract was centrifuged at 4° C., 6,500×g for 15 minutes, and then the supernatant was separated, added with ethanol in a volume 4 times (v/v) of the supernatant, and allowed to stand for 4 hours to precipitate polysaccharide. Dialysis (MWCO 6,000-8,000) was performed on the precipitated polysaccharide to obtain a crude polysaccharide fraction (BLW-0) extracted with hot water.

<Test Example>

Test Example 1. Analysis per composition of fractions

Constituent sugar analysis is performed by partially modifying the method of Albersheim et al. to derivatize each constituent sugar with alditol acetate and aldonolactone after hydrolysis, and then GC (Gas Chromatography ACME-6100, Young-Lin Co. Ltd. Anyang, Korea). ) Was used. The polysaccharide sample was hydrolyzed by reacting for 1.5 hr at 121° C. in 2 M TFA (trifluroacetic acid), dissolved in 1 ml of 1 M NH ₄ OH (ammonia solution), and reduced with 10 mg of NaBH ₄ for 4 hr. After adding an appropriate amount of acetic acid to remove the remaining NaBH ₄ , the mixture was repeatedly dried with methanol to remove excess acetic acid and converted to alditol corresponding to each constituent sugar. To find out the composition of neutral sugar, 1 ml of acetic anhydride was added to each alditol and reacted at 121° C. for 30 min to convert to alditol acetate, which was converted into chloroform/H ₂ O It was separated and extracted with a two-phase solvent system, and the extract was dried and dissolved in a small amount of acetone to be used as a sample for GC analysis.

In addition, in order to analyze the composition of acidic sugars, the converted alditol was dissolved in distilled water, developed on a Sep-pak QMA Cartridge (Waters, Milford, MA, USA), and aldonic acid was separated with 1 M HCl. 2 M TFA was added to the separated solution and reacted at 100° C. for 5 min to convert to aldonolactone. After that, it was separated and extracted with a chloroform/H ₂ O two-phase solvent system, dried and used as a GC analysis sample, and the GC column was an SP-2380 capillary column (0.25 ㎜×30 m, 0.2 ㎛ film thickness, Supelco, Bellefonte, PA , USA), a flame ionization detector (FID, Young-Lin Co. Ltd.) was used as the detector, and N ₂ as a carrier gas was flowed at 1.5 ml/min. Injection temperature of 250 ℃, detector temperature of 270 ℃, column temperature from 60 ℃ to 220 ℃ 30 ℃ / min, 220 ℃ to 250 ℃ under the conditions of 8 ℃ / min (see [Table 1]). The mole% of each component was calculated from the peak area ratio of each derivative, the reaction coefficient for the flame ionization detector (FID), and the molecular weight of the alditol acetate derivative of each component.

Apparatus GC ACME-6100 (Young-Lin Co. Ltd., Anyang, Korea) Detector Flame ionization detector(FID) (Young-Lin Co. Ltd., Anyang, Korea) Column SP-2380 capillary column (Supelco, Bellefonte, USA) Column size 0.25mm × 30m, 0.2m film thickness Oven temp. 60℃(1min) → 220℃(12min) → 250℃(15min) 30℃/min 8℃/min Injector temp. 250℃ Detector temp. 270℃ Carrier gas N ₂ (1.5mL/min)

As a result, as a result of analyzing the physicochemical properties such as the main component content and composition sugar composition of the hot water extract polysaccharide fraction (BLW-O) and RAPIDASE C80MAX (Pectinase) enzyme-treated polysaccharide fraction (BLE-0) from barley leaves, Table 2 below It was like.

Chemical composition
(Unit: wt%) Example 1 (BLE-0) Comparative Example 1 (BLW-0) Neutral sugar 74.77 ± 5.07 42.22 ± 4.60 Uronic acid 19.37 ± 0.47 44.64 ± 2.27 Protein 3.66 ± 0.53 11.54 ± 0.76 KDO-liked material 2.19 ± 0.22 1.60 ± 0.11 Composition of Sugar (Unit: mole%) Rhamnose 3.84 ± 0.04 2.60 ± 0.05 Fucose 0.60 ± 0.02 - Arabinose 16.56 ± 0.13 15.71 ± 0.20 Xylose 37.27 ± 0.07 5.39 ± 0.06 Mannose 1.15 ± 0.05 - Galactose 11.10 ± 0.11 14.78 ± 0.09 Glucose 4.25 ± 0.07 3.75 ± 0.13 GalA+GlcA 19.37 ± 0.47 44.64 ± 2.27

* In the table above, KDO means 2-keto-3-deoxy-D-manno-octulosonic acid and “GalA+GlcA” means the sum of galaturonic acid and glucuronic acid, and constitutes the uronic acid. .

* In the table above, the sugar composition was analyzed using the alditol acetates derivative method.

* The content ratio (%) of the chemical component represents the ratio in the dry sample.

* The mole% is calculated from the amount of total neutral sugar detected.

As a result of analysis of the general composition and constituent sugar of the polysaccharide fractions, Comparative Example 1 (BLW-0), which is a non-enzyme-treated polymeric polysaccharide fraction, contains an acidic sugar (42.22%) and acidic sugar (44.64%) at almost 1:1. Although shown as a polysaccharide fraction, Example 1 (BLE-0), which is an enzyme-treated polymer polysaccharide fraction, was analyzed as a neutral polysaccharide fraction in which the content of protein (3.66%) along with acidic sugar (19.37%) was rapidly reduced (Table 2).

This is due to the enzymatic action of the commercially available Rapidase ^TM , where pectinase is the main activity, and polygalacturonan [(α-D-galacturonic acid)n], which mainly comprises homogalacturonan (HG), was decomposed and removed by dialysis. In addition, in the composition of constituent sugars, Example 1 (BLE-0) mainly contains xylose, arabinose, and galactose (36.09, 16.04 and 10.74%), as well as a small amount of rhamnose, and thus rhamnogalacturonan I (RG-I) of pectins In addition to the arabinogalactan constituting ), the possibility of the existence of arabinoxylan, which is classified as hemicellulose, could be strongly deduced.

Test Example 2. In vitro anti-influenza efficacy test

In order to measure the antiviral effect on influenza virus H1N1, the following in vitro experiments were performed using a dog kidney cell line, MDCK (Madin-Darby canine kidney, ATCC: CCL-34). MDCK cells were placed in a 96 well microplate at 1×10 ⁵ /well per well, and cultured with medium EMEM (penicillin 100 units, streptomycin 100 μg, 10% FBS). When the MDCK cells became a monolayer, they were washed twice with EMEM medium containing only antibiotics. The H1N1 strain was diluted to 100TCID ₅₀ and placed in an EP tube, and the diluted polysaccharide material was added to each tube by concentration and then reacted at 4° C. for 1 hour. After 1 hour, the reaction solution was inoculated into pre-washed MDCK cells at 3 wells per concentration and incubated at 35° C. for 1 hour. After 1 hour, all the medium of the plate was removed, washed once with PBS, and 100 μl of the EMEM medium supplemented with antibiotics and 10 μg/mL trypsin was divided into each well and cultured at 37° C. for 48 to 72 hours. In the infection + non-administration control group, the cells were cultured for 48 to 72 hours until the cytopathic effect (CPE) appeared completely, and the cell status was observed daily with an inverted microscope. In general, the concentration at which the virus release is reduced by 50% compared to the control is EC50 (50% effective concentration), and the concentration at which the viability of cells is reduced by 50% is CC50 (50% cytotoxic concentration). The SI (selectivity index) is CC50/EC50, and the greater the SI value, the greater the virus proliferation inhibitory effect.

division CC50(mg/ml) ^a EC50 (mg/ml) ^b SI ^c Example 1 4.69±1.10 4.69±2.87 One Comparative example 9.32±2.10 15.69±1.79 0.59

As shown in Table 3 above, the enzyme-treated crude polysaccharide fraction (BLE-0) prepared according to Example 1 of the present invention has a larger SI value than the hot water-extracted crude polysaccharide fraction (BLW-0) of Comparative Example 1. It was confirmed that there is an effect of inhibiting virus proliferation.

Test Example 3. Measurement of Inhibitory Activity of Neuraminidase

The mechanism of the enzyme-treated crude polysaccharide fraction (BLE-0) for inhibiting influenza was studied. The Neuraminidase Inhibition Test is used as an index to measure antiviral activity against viruses that use the Neuraminidase (NA) protein system on the viral surface for viral proliferation, such as influenza virus. Neuraminidase activity inhibition test was performed according to the method recommended by WHO (Standard operating procedure WHO-025).

The virus to be used in this experiment was used as the IC ₅₀ of human-derived virus (H1N1), and enzyme inhibitory activity was investigated by concentration. MUNANA substrate (Methylumbellifery1)-α-DN-acetylneuraminic acid after reacting with each concentration sample solution and virus solution at room temperature for 45 minutes; sigma) was added, reacted at 37° C. for 1 hour, and then the stop solution was added, and then Ex. sigma) was added with a spectrofluorophotometer (ABI/Perkin Elmer Biosystems). 360nm/Em. Fluorescence was measured at 440 nm to investigate the inhibition of enzyme activity. The fluorescence value measured as a control without adding a sample was calculated as 100%.

[Equation 1]

Inhibitory activity (%) = [Sample + virus-treated group (OD) value-Sample + virus-free group (OD) value] / Sample + virus-untreated group (OD) value-Sample untreated + virus-free group (blank OD) value] × 100

division Example 1 (BLE-O) Comparative Example 1 (BLW-O) Inhibitory activity
(%) 0.01 mg/ml 10.0 2.7 0.1 mg/ml 20.7 12.0 1 mg/ml 30.9 21.2 10 mg/ml 81.2 30.5

As shown in Table 4 and FIG. 1 above, it was confirmed that the crude polysaccharide fraction prepared according to Example 1 of the present invention exhibits superior neuraminidase enzyme inhibitory activity compared to the crude polysaccharide fraction of Comparative Example 1.

This means that the crude polysaccharide fraction of barley leaves of Example 1 of the present invention can be used for the prevention or treatment of influenza virus infection diseases by inhibiting the activity of influenza virus.

2 is a diagram showing the inhibition of neuraminidase activity against various subtypes of influenza viruses (H3N2, H4N2, H5N2, H6N2, H7N2) derived from animals and humans of a crude polysaccharide fraction prepared according to Example 1 of the present invention. to be.

As shown in Fig. 2, the crude polysaccharide fraction prepared according to Example 1 of the present invention shows the inhibition of neuraminidase activity against all influenza viruses (H3N2, H4N2, H5N2, H6N2, H7N2) in a concentration-dependent manner. Confirmed.

Test Example 4. Animal test

Experimental animals

Balb/c mice 7 weeks old female were used, and safety was ensured in the case of the administered virus, but contact with other animals was blocked using a negative breeding system because of concerns about infection of other rodents. Isolation, identification, titer calculation and administration of the virus were performed in a special clean bench prepared to ensure its safety, and sterilization was performed before and after the experiment using a special disinfectant for virus disinfection. Feeding management, which can have a direct effect on body weight, was done by judging and feeding the amount of feed every day so that there were no restrictions on feeding. Each individual body weight was measured at a certain time on a weekly basis while controlling the factors that may affect the weight as much as possible. The determination of the effect on the weight gain by treatment group through comparison of the measured results for each individual was performed based on the average value of each treatment group. The lighting was changed every 12 hours, and water and food were freely consumed. Other matters related to animal breeding were conducted in accordance with the regulations of the National Association of Laboratory Animal Care, Chonbuk National University.

In order to evaluate the anti-influenza efficacy of Example 1, the fraction of Example 1 was administered orally for 14 days at each concentration (50 mg/kg/d, 100 mg/kg/d and 200 mg/kg/d). Influenza virus attack vaccination was performed by nasal passage with influenza virus H1N1 in a non-dose infection model (LD ₀ : 100% clinical symptoms, 0% mortality) after anesthesia using ether. The negative control used in this experiment was phosphate-buffered saline (PBS) administration + H1N1 non-infected group, and the virus control was phosphate-buffered saline (PBS) administration + H1N1 infection group.

-5 groups of mice-

Group 1 (negative control): PBS administration + H1N1 virus non-infection

Group 2 (virus control): PBS administration + H1N1 virus infection

Group 3 (BLE0-50): 50 mg/kg of crude polysaccharide fraction (BLE-0) + H1N1 virus infection

Group 4 (BLE0-100): crude polysaccharide fraction (BLE-0) 100 mg/kg administration + H1N1 virus infection

Group 5 (BLE0-200): crude polysaccharide fraction (BLE-0) 200 mg/kg administration + H1N1 virus infection

Test Example 4-1. Viral titer in the lungs

After the mice were sacrificed, the amount of virus remaining in the lungs of the mice was measured. The lung tissue of the mouse was separated and added to PBS for tissue emulsification, and 0.1 ml of the emulsion per egg was inoculated into the allantoic cavity of 10-day-old SPF eggs, and then placed in an incubator at 37° C. and cultured for 48 and 72 hours. After inoculation to at least 5 eggs per dilution and incubation for 48 and 72 hours, the virus infection titer was expressed as the reciprocal (Embryo infective dose; EID ₅₀ ) of the virus solution dilution that infects 50% of the chicken embryos. Calculated according to the method of Reed & Muench (Reed, L, J,. Muench, H.: A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493-497(1938)) .

As shown in Figure 3, the virus control group was confirmed that the EID ₅₀ significantly increased compared to the negative control group. In the case of the group to which the enzyme-treated crude polysaccharide fraction (BLE-0) was administered 200 mg/kg, it was confirmed that the EID ₅₀ decreased to a level similar to that of the negative control group.

Test Example 5. Reverse mutation toxicity test

In order to confirm the toxicity by the reversion mutation test of the crude polysaccharide fraction (BLE-0) prepared according to Example 1, the method suggested by Maron and Ames (1983) described in the literature was partially modified and tested as follows ( Maron, DM and Ames BN (1983) Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113: 173-215).

To search for mutagenicity for specific components, strains (Salmonella typhimurium) TA98, TA100, TA1535, and TA1537 were used. Treatment of the test substance was performed by a direct plate incorporation method in which metabolic active enzyme system (S-9 mix) was applied or not applied. Salmonella typhimurium TA98 strain used in the reversion mutation test was purchased from Molecular Toxicology Inc. (USA). For the strain, inoculate 50 μL of the frozen test strain solution into 25 mL of liquid medium (2.5% Oxoid Nutrient broth No. 2) and use a shaking incubator (VS-8480SFN, Vision Science Co., Ltd.). It was used after incubating for about 10 hours at ℃. The minimal glucose agar plate was prepared with 1.5% Bacto agar (214010, BD Difco), Vogel-Bonner medium E and 2% glucose, and the top agar was prepared with 0.6% agar and 0.5% NaCl. , 0.05 mM histidine (43011, Fluka)-biotin (47868, Supelco) was added to the top agar.

Dispense 2 mL each of the autoclaved top agar into a sterilized tube preheated to 45°C in a dry bath (11-718-4, Fisher Scientific), and 0.1 mL of the test substance solution and bacterial culture solution. 0.1 mL was mixed with a top agar, and immediately shaken with a vortex mixer (37600, Thermolyne) for 2-3 seconds, poured into a minimal glucose agar plate, tilted in various directions, and then solidified. For the excipient group (negative control), 0.1 mL of the excipient was added instead of the test material solution, and the positive control solution (2-Aminoanthracene (2AA), standard: Sigma A1381) was added in the same manner to the positive control group. After the top agar was hardened, the plate was turned over and incubated at 37° C. for about 48 hours with the plate lid closed, and then colonies were counted.

Strain Metabolic activity
Enzyme system Return mutant count/plate 0 μg/plate 500 μg/plate 1000 μg/plate TA98 -S9 20±1 23±0 18±4 +S9 24±3 24±1 21±0 TA100 -S9 135±6 140±8 121±6 +S9 145±4 134±5 142±6 TA1535 -S9 12±1 9±1 13±3 +S9 12±1 11±1 12±1 TA1537 -S9 12±1 10±1 11±1 +S9 14±1 9±1 11±2

As shown in Table 3 above, it was confirmed that the crude polysaccharide fraction (BLE-0) prepared according to Example 1 of the present invention did not induce a return mutation for the strain used under this test condition.

Test Example 6. In vitro staining abnormality test using Chinese Hamster Lung (CHL) cells

As an experiment to determine whether the crude polysaccharide fraction (BLE-0) prepared according to Example 1 causes structural or numerical abnormalities in the chromosomes of cultured Chinese Hamster Lung (CHL) cells, Chinese hamster lung (CHL) cells were used. Thus, a chromosomal abnormality test was performed with and without the application of the metabolic activity system. As a metabolic activity system, a cofactor was added to the rat liver homogenate induced by Aroclor-1254.

The test substance was suspended and treated in a physiological saline injection solution. The highest concentration was determined using Relative Increased Cell Count (RICC) as an index of cytotoxicity. The concentration group was set as shown in the following table, including the negative (excipient) control group and the positive control group, and one flask per concentration group was used.

Actively proliferating cells were isolated, 5 x 10 ⁴ cells were seeded in 5 mL of culture medium in a flask having a culture area of 25 cm ² , cultured for about 3 days, and the test material was treated. Chromosomal samples were prepared 24 hours after the start of treatment, and chromosomal abnormalities were counted from 150 metaphases per concentration group.

6 hours treatment with metabolic activity

Relative Increased Cell Count (RICC) was calculated by the following equation from the number of cells obtained by separating and counting cells from the flask, and used as an index of cytotoxicity.

[Equation 1]

S9 density
(μg/mL) Cell count RICC(%) Abnormal medium weather
(%) PP+ER(%) + 0 6388±123 100 0.00 0.00 31.25 6224±50 96 0.00 0.00 62.5 6297±139 98 0.00 0.00 125 5932±33 89 0.00 0.00 250 5943±17 89 0.00 0.00 500 6086±11 93 0.00 0.00 1000 5675±68 83 0.00 0.00 Benzo[a]pyrene 4418±246 52 34.00 0.00

Cloudiness was observed in the concentration group of 500 μg/mL or higher of the test substance. As shown in Table 4 above, the frequency of structural abnormal intermediate phase (excluding gap) was negative control, test substance 31.25, 62.5, 125, 250, 500 and 1000. In the order of μg/mL, 0.00, 0.00, 0.00, 0.00, 0.00, 0.00, and 0.00% showed no statistically significant increase in all concentration groups treated with the test substance compared to the negative control group, and there was no dose correlation. The incidence of mid-phase phase with a number of abnormalities was 0.00% in both the negative control group and all test substance treatment groups, and there was no statistically significant increase in all concentration groups treated with the test substance compared to the negative control group, and there was no dose correlation.

In the positive control group, a statistically significant increase was observed in the frequency of metaphase (34.00%) with structural abnormalities (P<0.01).

6 hours treatment without metabolic activation system

S9 density
(μg/mL) Cell count RICC(%) Abnormal medium weather
(%) PP+ER(%) - 0 8694±16 100 0.00 0.00 31.25 7894±55 88 0.00 0.00 62.5 7882±109 87 0.00 0.00 125 8097±7 91 0.00 0.00 250 8043±57 90 0.00 0.00 500 7963±109 89 0.00 0.00 1000 7206±3 77 0.00 0.00 4-Nitroquinoline-1-oxide 0.4 7306±26 78 12.00 0.00

As shown in Table 5 above, the frequency of structural abnormal intermediate phases is 0.00, 0.00, 0.00, 0.00, 0.00, 0.00 and 0.00% in the order of negative control, test substance 31.25, 62.5, 125, 250, 500, and 1000 μg/mL. , There was no statistically significant increase in all concentration groups treated with the test substance compared to the negative control group, and there was no dose correlation. The incidence of mid-phase phase with a number abnormality was 0.00% in both the negative control group and all test substance treatment groups, and there was no statistically significant increase in all concentration groups treated with the test substance compared to the negative control group, and there was no dose correlation.

In the positive control group, a statistically significant increase was observed in the frequency of metaphase (12.00%) with structural abnormalities (P<0.01).

24-hour treatment without metabolic activation system

S9 density
(μg/mL) Cell count RICC(%) Abnormal medium weather
(%) PP+ER(%) - 0 7642±51 100 0.00 0.00 31.25 7758±6 102 0.00 0.00 62.5 7527±3 98 0.00 0.00 125 7598±52 99 0.00 0.00 250 7227±44 92 0.00 0.00 500 6238±29 74 0.00 0.00 1000 5941±50 68 0.00 0.00 4-Nitroquinoline-1-oxide 0.4 6951±30 87 13.33 0.00

As shown in Table 6 above, the frequency of structural abnormal intermediate phases is 0.00, 0.00, 0.00, 0.00, 0.00, 0.00 and 0.00% in the order of negative control, test substance 31.25, 62.5, 125, 250, 500, and 1000 μg/mL. , There was no statistically significant increase in all concentration groups treated with the test substance compared to the negative control group, and there was no dose correlation. The incidence of mid-phase phase with a number abnormality was 0.00% in both the negative control group and all test substance treatment groups, and there was no statistically significant increase in all concentration groups treated with the test substance compared to the negative control group, and there was no dose correlation.

In the positive control group, a statistically significant increase was observed in the frequency of metaphase (13.33%) with structural abnormalities (P<0.01).

As a result of counting chromosomal abnormalities, the appearance frequency of the intermediate phase with chromosomal abnormalities in all test substance treatment groups did not increase compared to the negative control group, regardless of the treatment method, and this result did not satisfy the positive criteria.

Therefore, it was confirmed that the crude polysaccharide fraction (BLE-O), which is a test substance, does not cause chromosomal abnormalities in CHL cells under this test condition.

Hereinafter, examples of the formulation of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit this, but is intended to be described in detail.

Formulation Example 1. Preparation of powder

500 mg of crude polysaccharide fraction derived from barley leaves obtained in Example 1

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2. Preparation of tablet

300 mg of crude polysaccharide fraction derived from barley leaves obtained in Example 1

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3. Preparation of Capsule

200 mg of crude polysaccharide fraction derived from barley leaves obtained in Example 1

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

Formulation Example 4. Preparation of injection

600 mg of crude polysaccharide fraction derived from barley leaves obtained in Example 1

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above ingredients per ampoule according to a conventional injection preparation method.

Formulation Example 5. Preparation of liquid formulation

4 g of crude polysaccharide fraction derived from barley leaves obtained in Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add each ingredient to purified water to dissolve it, add lemon zest, mix the above ingredients, add purified water and add purified water to adjust the total to 100g, then fill in a brown bottle for sterilization. To prepare a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of crude polysaccharide fraction derived from barley leaves obtained in Example 1

Vitamin mixture right amount

Vitamin A acetate 70 ㎍

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

1.7 mg of nicotinic acid amide

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

1.75 mg ferrous sulfate

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules, but it is possible to arbitrarily modify the mixing ratio. After mixing the above ingredients according to a conventional granule preparation method, granules And can be used to prepare a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

1,000 mg of crude polysaccharide fraction derived from barley leaves obtained in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

Total 900 mL with purified water

After mixing the above ingredients according to a normal health drink manufacturing method, stirring and heating the resulting solution at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and stored in a refrigerator. It is used to prepare the functional beverage composition of the invention.

The composition ratio is composed of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences, such as the demand class, the country of demand, and the purpose of use.

Claims (19)

In the pharmaceutical composition for preventing or treating influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaf as an active ingredient,
The polysaccharide fraction comprises the steps of (a) treating barley leaf powder with pectin hydrolase; And (b) recovering the fraction by precipitating the enzymatically treated barley leaf powder with C1 to C4 alcohol; a pharmaceutical for the prevention or treatment of influenza virus infection disease, characterized in that it is prepared by a manufacturing method comprising Composition. The method of claim 1, wherein the barley leaf-derived polysaccharide fraction is 65 to 85% by weight of neutral sugar, 5 to 30% by weight of uronic acid, and 2-keto-3-deoxy (KDO) relative to the total polysaccharide fraction. -D-manno-octulosonic acid) comprising 0.5 to 10% by weight of a KDO-like substance consisting of a pharmaceutical composition for preventing or treating influenza virus infection diseases. According to claim 2, The barley leaf-derived polysaccharide fraction is a pharmaceutical composition for preventing or treating influenza virus infection disease, characterized in that it further comprises 0.5 to 20% by weight of protein. The pharmaceutical composition for preventing or treating influenza virus infection diseases according to claim 2, wherein the uronic acid is made of galacturonic acid and glucuronic acid. The pharmaceutical composition of claim 2, wherein the neutral polysaccharide comprises rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. According to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of influenza virus infection disease, characterized in that it inhibits neuraminidase activity. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits neuraminidase activity against H1N1, H3N2, H4N2, H5N2, H6N2 and H7N2. The pharmaceutical composition for preventing or treating influenza virus-infected diseases according to claim 1, wherein the disease caused by the influenza virus infection is at least one selected from the group consisting of flu, cold, sore throat, bronchitis and pneumonia. The pharmaceutical composition for preventing or treating influenza virus infectious diseases according to claim 8, wherein the flu is at least one selected from the group consisting of bird flu, swine flu and swine flu. delete The method of claim 1, wherein (c) recovering a fraction having a molecular weight of 30 kDa or more from the polysaccharide fraction of step (b); preventing influenza virus infection, characterized in that it is prepared by a method further comprising Or a therapeutic pharmaceutical composition. In the food composition for preventing or improving influenza virus infection disease containing a polysaccharide fraction derived from barley leaf as an active ingredient,
The polysaccharide fraction comprises the steps of (a) treating barley leaf powder with pectin hydrolase; And (b) recovering the fraction by precipitating the enzyme-treated barley leaf powder with C1 to C4 alcohol; a food composition for preventing or improving influenza virus infection, characterized in that it is prepared by a manufacturing method comprising . The method of claim 12, wherein the polysaccharide fraction derived from barley leaves is 65 to 85% by weight of neutral sugar, 5 to 30% by weight of uronic acid, and 2-keto-3-deoxy (KDO) relative to the total polysaccharide fraction. -D-manno-octulosonic acid) KDO-like material consisting of 0.5 to 10% by weight of a food composition for preventing or improving influenza virus infection, characterized in that it comprises 0.5 to 10% by weight. The food composition for preventing or improving influenza virus infectious diseases according to claim 12, wherein the food composition inhibits neuraminidase activity. The food composition of claim 12, wherein the food composition inhibits neuraminidase activity against H1N1, H3N2, H4N2, H5N2, H6N2 and H7N2. In the pharmaceutical composition for animals other than humans for the prevention or treatment of influenza virus infection disease containing a polysaccharide fraction derived from barley leaf as an active ingredient,
The polysaccharide fraction comprises the steps of (a) treating barley leaf powder with pectin hydrolase; And (b) recovering the fraction by precipitating the enzyme-treated barley leaf powder with C1 to C4 alcohol; a human for the prevention or treatment of an influenza virus infection disease, characterized in that it is prepared by a manufacturing method comprising Except for animal pharmaceutical compositions. The method of claim 16, wherein the animal influenza virus infectious disease is mutant infectious bird flu, Avian paramyxoovirus infection due to newcastle disease virus, Canine distemper infection, chicken infectious bronchitis Turkey coronavirus infection due to virus (infectious broncheitis virus), avian pneumovirus infection due to avian pneumovirus, pig genital tract due to porcine reproductive and respiratory syndrome virus, and Respiratory syndrome virus infection, Foot-and-mouth disease due to coxsakie virus, Avian leukosis due to reticuloendotheliosis virus, Avian leukosis due to aujeszky's disease virus Chicken infectious laryngotrachitis, respiratory infections caused by adenovirus, and animal adenovirus infections that cause egg drop syndrome, characterized by at least one selected from the group consisting of influenza virus infections. Pharmaceutical compositions for animals other than humans for the prevention or treatment of diseases. In the feed composition for preventing or improving influenza virus infection disease containing a polysaccharide fraction derived from barley leaf as an active ingredient,
The polysaccharide fraction comprises the steps of (a) treating barley leaf powder with pectin hydrolase; And (b) recovering the fraction by precipitating the enzyme-treated barley leaf powder with C1 to C4 alcohol; a feed composition for preventing or improving influenza virus infection disease, characterized in that it is prepared by a manufacturing method comprising . In the feed additive composition for preventing or improving influenza virus infectious diseases containing a polysaccharide fraction derived from barley leaf as an active ingredient,
The polysaccharide fraction comprises the steps of (a) treating barley leaf powder with pectin hydrolase; And (b) precipitating the enzyme-treated barley leaf powder with C1 to C4 alcohol to recover the fraction; feed additive for preventing or improving influenza virus infection disease, characterized in that it is prepared by a manufacturing method comprising Composition.

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