KR20130081478A - A composition comprising the ivy leaf extract and the compound isolated therefrom for preventing and treating influenza viral disease - Google Patents

Abstract

PURPOSE: A hedera helix leaf extract or a compound separated from the same are provided to utilize as a medicinal composition for prevention and treatment of an infectious diseas by an influenza virus or a health functional food by checking a high survival rate when taken together with an antivirus agent. CONSTITUTION: A medicinal composition for prevention and treatment of an infectious disease caused by an influenza virus contains a Hedera helix leaf extract or hederacoside C separated from the same as an active ingredient. The Hedera helix leaft extract is a crude extract, a polar solvent soluble extract, and a non-polar solvent soluble extract. The medicinal moposition for prevention and treatment of an infectious disease contains the combination of the Hedera helix leaf extract or hederacoside C and an anti-influenza agent. The anti-influenza agent is selected from Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel), Rimantadine, Rivaririn, or Taribavirin. [Reference numerals] (AA) Survival rate; (BB) Medicine injection; (CC) Time (day) after infection; (DD) Extract of Ivy leaf

Description

A composition comprising the ivy leaf extract and the compound isolated there from for preventing and treating influenza viral disease}

The present invention provides a composition for the prevention and treatment of influenza virus-related diseases containing an ivy leaf extract and a compound isolated therefrom as an active ingredient.

Selmons et al., Avian Dis., 18 (1), p. 119-124, 1974.

Webster RG et al., Microbiol Rev., 56 (1), p. 152-179, 1992.

Document 3 Alexander DJ, Vet. Microbiol., 74 (1-2), p. 3-13, 2000.

Document 4 Walter JH, Bull. NY Acad. Med., 54, p. 855-864, 1978.

[5] Taubenberger JK et al., Science, 275, p. 1179-1796, 1997.

6 Potter CW, J. appl. Microbiol., 91, p. 572-579, 2001.

[Reference 7] Oxford JS, Rev. Med. Virol., 10 (2), p. 119-133, 2000.

Capua I et al., Avian Pathol., 32, p. 47-55, 2003.

9 Capua I and Marangon S, Avian Pathol., 32, p. 335-343, 2003.

Document 10 Ward P et al. J. antimicrob. Chemother., 55 (supp1), p.i5-i21, 2005.

[Document 11] Korean Patent Publication No. 10-2004-106201.

12 Demirci et al., Pharmazie 59, 770-774, 2004.

Document 12. Korean Patent Publication No. 10-2006-8310.

Document 13 Majester-Savornin et al., Planta Med. 57, 260-262, 1991.

Document 14 Exp. Parasitol. 116, 340-345, 2007.

15 Mendel et al., J. Ethnopharmacol. 134, 796-802, 2011.

16 Gepdiremen et al., Phytomedicicne 12, 440-444, 2005.

[Document 17] Panov etc., Triterpene glycosides from Kalopanaxseptmlobum .Ⅵ. Glycosides from leaves of Kalopanax septemlobum var. typicum introduced to crimea, Chemistry of Natural Compounds , 42, 49-54 (2006).

[Document 18] Panov etc., Triterpene glycosides from Kalopanaxseptmlobum .Ⅶ. Minor glycosides from stems of Kalopanax septemlobum var. maximowiczii and Kalopanx septemlobum var. typicum , Chemistry of Natural Compounds , 42, 61-66 (2006).

[19] Taskin et al., Triterpene saponins from Nigella sativa L., Turkish Journal of Chemistry , 29, 561-569 (2005).

20 Sang-Uk Seo et al., J. Virol. 2010. 84 (24): 12713-12722.

Due to the concentration of the population due to the development and urbanization of transportation, respiratory infectious diseases caused by influenza, pneumococci, etc., which are spread to the air, pose a serious threat to the public. Among them, influenza virus belongs to the orthomyxovirus family and is an RNA virus that is infected through the respiratory mucosa. The glycoproteins HA (Hemagglutinin) and NA (Neuraminidase) are present on the surface and are classified into various subtypes according to the antigenicity of HA and NA. . In addition, Avian influenza A (H5N1), the first human infection reported in 1997, continues to report infections in livestock, including birds, with a mortality rate of more than 60%. It is the object of alert.

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and serotypes are classified into three types, type A, type B and type C. Among them, hepatitis B and C have been identified only in humans, while hepatitis A has been identified in humans, horses, pigs, other mammals and various types of poultry and wild birds (Selmons et al., Avian Dis. , 18 (1), p. 119-124, 1974; Webster RG et al., Microbiol Rev., 56 (1), p. 152-179, 1992).

The serotypes of influenza A viruses are classified according to the two types of proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA), and 144 types according to the serotype. 16 species and 9 NA types). The HA protein plays a role in attaching the virus to somatic cells, and the NA protein allows the virus to penetrate into the cell (Alexander DJ, Vet. Microbiol., 74 (1-2), p.3-13, 2000). The normal natural host of influenza A virus is known as wild waterfowl such as ducks and seagulls.As a result of epidemiological investigations of influenza infection in wild birds around the world, there are 16 existing HA and 9 NA influenza species. The virus was confirmed to be infected in wild birds (Selmons et al., Avian Dis., 18 (1), p. 119-124, 1974). Avian influenza virus, classified as type A, is a common infectious disease virus, a non-pathogenic avian influenza that causes mild respiratory symptoms when infected with chickens, and low pathogenicity that causes mortality and spawning degradation of about 1-30%. Low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) with more than 95% high mortality and also called "bird flu" (Alexander DJ, Vet. Microbiol., 74 (1-2), p. 3-13, 2000). The highly pathogenic algal influenza is classified as Class A by the International Water Services Bureau (OIE) and domestic type 1 infectious disease.

The first record of human influenza dates back to 412 B.C.E., but the first pandemic influenza record of humanity is estimated to have occurred throughout Europe from 1173 to 1174. Since the 20th century, records of influenza have been more scientifically treated, based on the data that have been left behind, since the 20th century there have been three pandemic influenza. Since the twentieth century, a worldwide pandemic between 1918 and 1920, the first influenza pandemic (aka Spanish flu) has been documented as the greatest damage to humanity, with between 20 and 50 million people dying during that period ( Walter JH, Bull.NY Acad.Med., 54, p. 855-864, 1978). The causative agent of the Spanish flu virus has been identified as serotype A / H1N1, which is very similar to the swine influenza virus and is still a major epidemic of pandemic flu, which is still prevalent worldwide every year (Taubenberger JK et al., Science, 275, p.1793-). 1796, 1997). Since the 20th century, which occurred between 1957 and 1958, the second influenza pandemic began in China and rapidly spread along the coast to nearby Hong Kong, Singapore, Japan and Taiwan in six to seven months. About 40-50% of the world's population was infected during this period, of which 25% had clinical symptoms, mainly infants and middle-aged people who died of infection, and about 1 million people died. (Potter CW, J. appl. Microbiol., 91, p.572-579, 2001). The causative virus was identified as serotype A / H2N2 influenza virus. In addition, the third pandemic of the influenza pandemic between 1968 and 1969 was confirmed to originate in Hong Kong and spread rapidly to Taiwan, the Philippines, Singapore and Vietnam. The causative virus is serotype A / H3N2, which differs from the previous serotype H2N2 virus and HA, which has been analyzed to be transmitted from birds, and is still the main epidemic of pandemic flu, which is still prevalent worldwide every year. (Oxford JS, Rev. Med. Virol., 10 (2), p. 119-133, 2000).

Influenza viruses can infect the respiratory tract, cause systemic symptoms, change their appearance periodically, and move to another host before the host dies, without killing the host. Although it is the virus that causes the greatest economic loss to human beings and preventive vaccines have been developed, they have not caught up with the mutation of the virus, and the basic virus treatment is not yet performed.

Among the avian influenza vaccines, live virus vaccines are almost impossible to develop due to the characteristics of viruses that are easily mutated. The vaccines developed to date can be largely classified into a deadly poison vaccine and a recombinant vaccine. As HPAI outbreaks prolonged and spread throughout the country in 1999 and Hong Kong in 2003, avian influenza vaccine was used as a means of combating HPAI in combination with a selective killing policy. Currently, in Italy and Hong Kong, the use of avian influenza vaccine (Capua I et al., Avian Pathol., 32, p.47-55, 2003; Capua I and Marangon S, Avian Pathol., 32, p.335-343, 2003). Vaccines positively evaluated in Italy (serum type A / H7N1) and Hong Kong (serum type A / H5N1) are all serotoxin vaccines and are heterologous serotypes with the same HA type but different NA type (serum type A / H7N3, A serotoxin vaccine with serotype A / H5N2) was used to distinguish it from field infection in antibody testing. However, the deadly venom vaccine cannot be distinguished from vaccine and field infection by the Agar Gel Precipitation (AGP) test, which is the standard A type of avian influenza standard diagnosis. It is pointed out that it is not suitable for inspection. In addition, the currently developed dead venom vaccine can reduce the amount of virus released into feces during HPAI infection, but does not completely prevent the spread of disease (Capua I et al., Avian Pathol., 32, pp 47-55, 2003; Capua I and Marangon S, Avian Pathol., 32, p. 335-343, 2003).

Amantadine and rimantadine are substances that inhibit the function of M2 ion channel proteins of influenza viruses and are representative antiviral agents that inhibit the growth of influenza viruses in vivo. However, these two antiviral agents were found to be effective only against serotype A influenza virus and not against serotype B influenza virus without M2 protein. 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 influenza virus M2 protein when used. Zanamivir and oseltamivir, which are developed to compensate for these drawbacks, inhibit the function of the neuraminidase protein of influenza viruses and are representative of the suppression of influenza virus in vivo. Antiviral agents. These two antiviral agents are known to be effective against all 16 serotype A influenza viruses and serotype B influenza viruses. However, zanamivir has the disadvantage of inhalation and intravenous administration, while oseltamivir can be administered orally, but it has been pointed out as a disadvantage due to side effects such as recent reports of resistant virus and vomiting and dizziness upon oral administration (Ward P et. al., J. antimicrob.Chemother., 55 (supp1), p.i5-i21, 2005). Therefore, along with vaccines and therapeutics, the development of safe natural materials while increasing human immunity will be an important means of reducing mortality during pandemic.

To date, subunit vaccines, inactivated vaccines or attenuated vaccines, etc. have been used to prevent influenza virus infections, but the prevention of the influenza virus is limited due to high antigenic mutations. For example, influenza A (H1N1), first detected in March 2009 in San Diego, California, USA, affected more than 14,000 people in 48 countries, including Korea, in just two months. It is epidemic worldwide and has resulted in more than 130,000 infections and more than 300 deaths ( http://www.who.int/influenza ).

Accordingly, antiviral agents currently used to treat influenza infections include Oseltamivir (Tamiflu) and Zanamivir (Relenza), which are inhibitors of influenza NA (neuraminidase), or Amantadine and Rimantadine, which are inhibitors of M2 ion channels of influenza A virus. It is used for the purpose of prevention and treatment of influenza, and it has been reported to be effective to administer influenza virus using these antiviral agents within 24-48 hours after infection. In particular, the recent emergence of resistant viruses to these antiviral agents is the urgent situation to develop a new concept of treatment to overcome this.

On the other hand, Ivy ( Hedera helix ) is a plant called English ivy, the leaf extract has an expectorant action and is used for the treatment of bronchitis (Korean Patent Publication No. 10-2004-106201), its main ingredient is saponin called hederacoside (Demirci et al., Pharmazie 59, 770-774, 2004: Korean Patent Publication No. 10-2006-8310). In addition to their expectorant action, the extracts of their leaves have been reported to have antibacterial, antifungal, anti-lesmanian, and digestive effects (Majester-Savornin et al., Planta Med. 57, 260-262, 1991; Exp. Parasitol. 116, 340-345, 2007, Mendel et al., J. Ethnopharmacol. 134, 796-802, 2011), several activities of hederacoside C, the active substance of ivy leaf, have been reported. It is. In other words, anti-inflammatory activity, antibacterial activity and antifungal activity have been reported (Gepdiremen et al., Phytomedicicne 12, 440-444, 2005), but there is no known antiviral activity.

Neither of these documents discloses or teaches the inhibitory activity of ivy leaf extracts and compounds isolated therefrom against influenza viruses.

Therefore, the present inventors have tested the inhibitory activity against influenza virus against ivy leaf extract and Hederacoside C isolated from it for the purpose of use as a therapeutic agent for influenza virus infection, showing a strong inhibitory activity. It was found for the first time that high activity was exhibited especially when combined with existing antiviral agent, and completed this invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of infection by influenza virus containing an ivy leaf extract and hederacoside (Hederacoside) C separated therefrom as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of the infection by influenza virus containing an ivy leaf extract and hederacoside (Hederacoside) C separated therefrom as an active ingredient.

Ivy leaf extract as defined herein is characterized in that the crude extract, polar solvent soluble extract or non-polar solvent soluble extract.

The crude extract as defined herein is a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol and butanol, or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably 30 to 30 Characterized in that the extract is available in 100% ethanol.

Non-polar solvent soluble extract fractions as defined herein include extract fractions soluble in non-polar solvents purified only from the crude extract of the present invention in hexane, methylene chloride, chloroform, or ethyl acetate, preferably hexane, or chloroform solvent. do.

The polar solvent-soluble extract as defined herein is prepared by removing the non-polar solvent-soluble fractions from the crude extract and washing with water, methanol, butanol or a mixed solvent thereof, preferably water or butanol, more preferably, And one extracted fraction.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of infection by influenza virus containing a combination of ivy leaf extract, or Hederacoside (Hederacoside) C isolated from it and an existing antiviral agent as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of the infection by influenza virus containing a combination of ivy leaf extract, hederacoside C isolated from it and a conventional antiviral agent as an active ingredient. .

Existing antiviral agents as defined herein are Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel) and Rimantadine, Rivaririn, Taribavirin, and the like, preferably Oseltamivir or Amantadine.

Influenza viruses as defined herein are H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 or H10N2 or H10N7 It is characterized by having the H1N1 serotype.

Infections caused by influenza viruses as defined herein include avian influenza (bird flu), pandemic flu, and flu.

The compounds of the present invention may be prepared into pharmaceutically acceptable salts and solvates by methods conventional in the art.

Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be filtered by suction.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, and hydroiodic acid.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate rate, methane sulfonate (mesylate) and p - toluene sulfonate (tosylate) and a salt, the salt manufacturing method or manufacturing process known in the art ≪ / RTI >

Hereinafter, the present invention will be described in more detail.

The extracts and compounds of the present invention can be obtained by the following production methods.

For example, the present invention will be described in detail below.

Ivy leaf extract of the present invention can be prepared as follows. After washing and cutting the dried ivy leaf, a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably Ultrasonic extraction, hydrothermal extraction, room temperature extraction for 30 minutes to 48 hours, preferably 1 hour to 12 hours at a temperature of 30 ° C. to 150 ° C., preferably 50 ° C. to 100 ° C. Alternatively, the crude extract of the present invention may be obtained by filtering, concentrating under reduced pressure, and drying the extract obtained by reflux extraction, preferably by performing ultrasonic extraction about 1 to 20 times, preferably 2 to 10 times.

In addition, the polar solvent or non-polar solvent soluble extract of the present invention is about 0.0005 to 0.005 times the weight of the crude extract, preferably 30 to 90% ethanol crude extract, preferably 0.05 to 0.5 times the volume (v / w% Non-polar solvent soluble extract fractions which are available in non-polar solvents such as n-hexane, methylene chloride, ethyl acetate by carrying out a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol after addition of water; And polar solvent-soluble extract fractions soluble in polar solvents such as butanol and water can be obtained.

Flash column using a method of increasing the polarity of the hexane and methylene chloride mixed solvent in a non-polar solvent soluble extract fraction, preferably a hexane soluble fraction obtained in a non-polar solvent such as n-hexane, methylene chloride, ethyl acetate obtained above Purification methods using chromatography, such as chromatography, RP C18 column chromatography, or silica gel open column chromatography, may be optionally repeated several times, thereby purifying and obtaining Hederacoside C, a compound of the present invention, respectively. .

The present inventors have tested the inhibitory activity against the influenza virus of the extracts and compounds obtained by the above production method, showed a strong inhibitory activity, especially when combined with the existing antiviral agent shows a high virus inhibitory activity By confirming, the composition was found to be useful as a pharmaceutical composition or health functional food for the prevention and treatment of infection by influenza virus.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of infection by influenza virus containing the ivy leaf extract obtained by the above method, a compound isolated therefrom as an active ingredient.

In addition, the ivy leaf is a drug that has been used for a long time as an edible or herbal medicine isolated from the ivy leaf extract of the present invention also has no problems such as toxicity and side effects.

The pharmaceutical composition of the present invention contains 0.1 to 50% by weight of the above extract or compound, based on the total weight of the composition.

The pharmaceutical compositions comprising the extracts or compounds of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Pharmaceutical dosage forms of the extracts or compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

The composition comprising the extract or the compound of the present invention may be administered orally or topically in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And can be used as formulations.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules and capsules, which may contain at least one excipient such as starch, calcium carbonate, sucrose, ), Lactose, gelatin and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyloleate may be used. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, and glycerol gelatin can be used.

The preferred dose of the extract or compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention may be administered in 0.0001 ~ 100 mg / kg, preferably in an amount of 0.001 ~ 100 mg / kg divided once to several times daily. The compound of the present invention in the composition may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural and intracere broventricular injections.

The present invention also provides a dietary supplement for the prevention and improvement of the infection caused by influenza virus containing an ivy leaf extract obtained by the above method, or a compound isolated therefrom as an active ingredient.

Extracts or compounds of the present invention can be used in a variety of drugs, food and beverages for the prevention and treatment of the desired disease. Foods to which the extract or compound of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes and dietary supplements, and are in the form of powders, granules, tablets and capsules or beverages. Can be used.

The extract or the compound of the present invention may be added to a food or beverage for the purpose of prevention and treatment of a target disease. At this time, the amount of the extract or compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Preferably at a rate of 0.3 to 10 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like , Xylitol, sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract or the compound of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the extract or the compound of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As mentioned above, the ivy leaf extract of the present invention or a compound isolated therefrom showed a significant increase in survival as a result of measuring the survival rate of mice in an influenza virus infected animal model of the extract and compounds. In particular, by confirming the high survival rate increase in combination with the existing antiviral agent, the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of infection by influenza virus.

1 is a diagram showing the survival rate of mice infected with influenza virus of 30% ethanol ivy leaf extract;
2 is a diagram showing the survival rate of mice infected with influenza virus by Osteltamivir administration;
3 is a diagram showing the survival rate of mice infected with influenza virus by the combination of ivy leaf extract and Oseltamivir;
Figure 4 is a diagram showing the survival rate of influenza virus infected mice by the combination of ivy leaf extract fraction and Oseltamivir.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Reference Examples and Experimental Examples.

Example  One. Ivy leaves  Preparation of extract

1-1. Ivy leaves Crude extract  Produce

600 g of dried leaves of English Ivy leaf, identified under Professor Kwon Yong-soo (Natural Products, yskwon@kangwon.ac.kr), Kangwon National University, were mixed with various concentrations of water and ethanol, that is, 30% and 200 ml of 70% (v / v) ethanol solvent was added, extracted three times at 60 ° C. with an ultrasonic extractor (8510 DHT; CT, USA) for 3 hours, and then vacuum filtered to recover the supernatant. This process was repeated four times, and the supernatant was collected, and then dried under reduced pressure and lyophilizer (FD5512, Ilshin, Seoul, Korea) to extract 30% and 70% (v / v) ethanol mixed solvent extracts of the ivy leaf, respectively. g and 80.2 g were obtained respectively (hereinafter referred to as "HH30E" and "HH70E", respectively).

In addition, 30% and 70% ethanol extract of the ivy leaf was confirmed to be the same when compared with the extract purchased from Europe imported Sinpung Pharmaceuticals.

1-2. Polar solvent and Nonpolar solvent  Fraction of soluble extract

The dried 30% ethanol extract (50 g) obtained in Example 1-1 was suspended in distilled water, and n-hexane, chloroform and n-butolol (BuOH) were prepared according to a conventional fractionation method in the art. ), And dried under reduced pressure to obtain n-Hexane soluble fraction (2.2 g; hereinafter referred to as "HH-H"), chloroform soluble fraction (0.7 g; referred to as "HH-C"), n-BuOH The soluble fraction (10.0 g; hereinafter referred to as "HH-B") and the water residue were lyophilized to give 21.0 g (hereinafter referred to as "HH-W").

Example   2. Ivy leaves  Isolation of Active Compounds from Extracts

In order to identify the active substances, in the following experiment, water residue (10 g) of the most active fractions was placed in a silica gel column and CHCl ₃ : MeOH: water (7: 5: 1) was added to the mobile phase. Chromatography was carried out, and this separation was repeated to give hederacoside C (hederagenin-3-O-α-L-rhamanopyranosyl (1 → 2) -α in the form of amorphous powder white having the following physical properties. -L-arabinopyransyl28-O-α-L-rhamnopyranosyl (1 → 4) -β-D-glucopyranosyl (1 → 6) -β-D-glucopyranoside ( see Structural I ) (420 mg) was obtained, which was obtained as a reference ( Compared with Engelhard Arzneimittel GmbH & Co. Germany) and the existing literature data, the structure was confirmed to be identical (Panov et al., Triterpene glycosides from Kalopanaxseptmlobum.VI.Glycosides from leaves of Kalopanax) septemlobum var. typicum introduced to crimea, Chemistry of Natural Compounds , 42, 49-54 (2006); Panov, etc., Triterpene glycosides from Kalopanaxseptmlobum .Ⅶ. Minor glycosides from stems of Kalopanax septemlobum var. maximowiczii and Kalopanx septemlobum var. typicum , Chemistry of Natural Compounds , 42, 61-66 (2006); Taskin et al., Triterpene saponins from Nigella sativa L., Turkish Journal of Chemistry , 29, 561-569 (2005)].

(I)

¹ H-NMR (600 MHz, pyridine- d ₅ ): 6.24 (1H, d, J = 8.4 Hz, Glu-1 '''), 6.23 (1H, brs, Rham-1''), 5.86 (1H, brs , Rham-1 '''''), 5.13 (1H, d, J = 6.1 Hz, Ara-1'), 5.00 (1H, d, J = 8.1H, Glu-1`` ''), 1.71 ( 3H, d, J = 6.2 Hz, Rham-6 '''''), 1.65 (3H, d, J = 6.2 Hz, Rham-6``), 1.18 (3H, s, 27-CH ₃ ), 1.13 (3H, s, 26-CH ₃ ), 1.08 (3H, s, 24-CH ₃ ), 0.99 (3H, s, 25-CH ₃ ), 0.89 (3H, s, 30-CH ₃ ), 0.87 (3H , s, 29-CH ₃ );

¹³ C-NMR (125 MHz, pyridine- d ₅ ): 176.6 (C-28), 144.1 (C-13), 122.9 (C-12), 104.9 (Glu-1`` ''), 104.3 (Ara-1 '), 102.8 (Rham-1'''''), 101.7 (Rham-1 ''), 95.7 (Glu-1 '''), 81.1 (C-3), 78.7 (Glu-3''') , 78.3 (Glu-4 '''', Glu-5 '''), 78.1 (Glu-3''''), 77.2 (Glu-5''''), 76.5 (Glu-2''') , 75.9 (Arb-2 '), 75.4 (Glu-2''''), 74.2 (Rham-4''), 74.0 (Rham-4'''''), 73.9 (Ara-3 '), 72.8 (Rham-3 ''), 72.6 (Rham-2 ''''',Rham-3'''''), 72.0 (Rham-2 ''), 70.9 (Glu-4 '''), 70.3 ( Rham-5 '''''), 69.8 (Rham-5''), 69.3 (Glu-6'''), 69.2 (Ara-4 '), 65.6 (Arb-5'), 64.0 (C-23 ), 61.3 (Glu-6`` ''), 48.2 (C-9), 47.7 (C-5), 47.1 (C-17), 46.2 (C-19), 43.5 (C-4), 42.2 ( C-14), 41.7 (C-18), 39.9 (C-8), 39.1 (C-1), 36.9 (C-10), 34.0 (C-21), 33.1 (C-29), 32.8 (C -7), 32.6 (C-22), 30.8 (C-20), 28.3 (C-15), 26.2 (C-2), 26.1 (C-27), 23.9 (C-11), 23.7 (C- 30), 23.4 (C-16), 18.5 (Rham-6``, Rham-6 '''''), 18.2 (C-6), 17.6 (C-26), 16.2 (C-25), 14.0 (C-24).

Experimental Example  1. Inhibitory activity against influenza virus

In order to confirm the inhibitory activity against the influenza virus of the samples obtained in the above example, the experiment was performed by applying the method disclosed in the literature (Sang-Uk Seo et al., J. Virol. 2010. 84 (24) 1271-12722).

1-1. Influenza virus  culture

In order to obtain influenza virus A / PR / 8/34 (PR8, H1N1) from eggs, SPF fertilized eggs (orientbio) were grown for 11 days in a 37 ° C incubation period and then labeled 2/3 site to the lateral vesicle of the fertilized egg. . The A / PR / 3/34 virus parent strain was diluted 10 ⁴ to 10 ⁶ times by a step dilution method using PBS (Phosphate-Buffered Saline) to infect 100 ul using a syringe. The infected area was closed with a paraffin to prevent infection by other bacteria or viruses. Infected fertilized eggs were grown in a 37 ° C. incubator for 3 days and then stored at 4 ° C. for 12 hours. Urinary fluid was collected after cutting the bladder area with surgical scissors. The ureter was passed through a filter and then dispensed and stored at -80 ° C. For virus titer, 10 ⁶ MDCK cells (Canine kidney epithelial, ATCC) were passaged in each well of a 6 well plate. The next day, virus samples were diluted by step dilution using MEM medium (Invitrogen) and 500 ul of each well was added. Infection was performed on a rocking plate at room temperature for 30 minutes, and after infection, cells were washed with MEM medium and then cultured for 2 days by adding 5 ug / ml of trypsin (Invitrogen) to MEM medium containing 1% agarose. . After 2 days, the number of plaques was measured in a well where 50-200 plaques were observed, and the titer of the virus sample was determined by inverting the dilution factor.

1-2. Influenza virus Dosage and observation of symptoms

The mice used in the experiment (C57BL / 6, female, 6 weeks, purchased from Orient-Bio) were intraperitoneally mixed with ketamine (100 mg / kg, Yuhan) and lump (20 mg / kg, BAYER) before infection. Anesthetized by administration. The nasal cavity of anesthetized mice was infected three times with virus diluted 1 × 10 ⁵ pfu at 30 ul. Survival of mice assessed resistance to influenza virus.

In order to verify the anti-influenza effect of ivy leaf extract and fractions in the influenza infection model, 10 ⁵ pfu / 30 ul of PR8 influenza virus was intranasally administered to mice, and the extracts or fractions of the examples were administered orally for 5 days 2 days after influenza administration. Afterwards (30 mg / Kg) mouse survival was observed.

1-3. Ivy leaves  The extract and Fraction  Antiviral effect

As a result of the experiment, when the oral administration of 30% ethanol extract was observed a significant increase in survival compared to the control mice (Fig. 1). Therefore, it was confirmed that the anti-influenza effect of the mouse significantly increased by administration of ivy leaf extract.

In a similar manner, the influenza treatment effect of the 30% or 70% extract of the ivy leaf and the fraction obtained in the example is shown in Table 1.

Antiviral Effects of Ivy Leaf Extracts and Fractions Group Dose (mg / Kg) n 15 days after infection
Survival rate (%) PR8-treated - 20 0 PR8 + Ivy Lobe 30% EtOH Extract (30mg / Kg) 30 20 15 PR8 + Ivy Leaf 70% EtOH Extract (30mg / Kg) 30 20 5 Hexane fraction of PR8 + 30% EtOH extract 30 10 0 CHCl ₃ fraction of PR8 + 30% EtOH extract (30 mg / Kg) 30 10 0 NBuOH fraction (30 mg / Kg) of PR8 + 30% EtOH extract 30 10 10 Water residue (30mg / Kg) from PR8 + 30% EtOH extract 30 10 20

1-3. Antiviral effect by coadministration with existing antiviral agents

In the case of antiviral agents currently used for influenza viruses, the emergence of antiviral resistant influenza has been reported, and therefore measures to overcome them are urgently needed. Therefore, we tried to increase the effect of anti-influenza when used in combination with anti-influenza, which is currently being used, to prevent the emergence of resistant virus and to determine whether it can be used in combination with influenza treatment. Prior to this, anti-influenza effects according to the dose of Osteltamivir (Sigma), the most widely used anti-influenza drug, were evaluated in a mouse model. After nasal administration of 10 ⁵ pfu of PR8, 2 days later, 1, 5, 25 mg / kg of Oseltamivir was orally administered for 5 days, and the survival rate of the mice was observed.

The mice treated with 25 mg / kg of Osteltamivir showed 100% survival rate and the mice with oral administration of 5 mg / kg of Oseltamivir showed 50% survival rate (FIG. 2). Therefore, the effect of the combination administration of the ivy leaf extract on mice receiving 5 mg / kg Oseltamivir, which is a dose similar to that used in humans (75 mg tablet x 2 times / day), based on body weight, was evaluated.

In other words, to determine whether the administration of ivy leaf extract can increase the antiviral effect of Oseltamivir, the mice were infected with 10 ⁵ pfu of influenza virus, followed by oral administration of ivy leaf extract and Oseltamivir once a day for 5 days. It was administered in combination. 60% of mice survived with Oseltamivir (5mg / Kg) alone, compared with Oseltamivir alone, which showed a 100% survival rate compared with Oseltamivir alone (Fig. 3). ). Therefore, it was confirmed that the ivy leaf extract not only has anti-influenza effect alone but also shows synergistic anti-influenza activity when administered in combination with other existing anti-influenza therapeutics such as Oseltamivir.

In addition, the fractions and residues separated in Example was confirmed that the significant anti-influenza effect by water residue when co-administered with the antiviral agent (Fig. 4).

In a similar manner, the antiviral effect of the ivy leaf extract and fractions and the co-administration effect with the existing antiviral agents are shown in Table 2.

Combination Administration of Ivy Leaf Extract with Antiviral Agents Group Volume
(mg / Kg) n 15 days after infection
Survival rate (%) PR8-treated - 9 0 PR8 + Oseltamivir (5mg / Kg) 30 10 60 Hexane fraction of PR8 + Oseltamivir (5mg / Kg) + 30% EtOH extract 30 10 60 CH8 ₃ fraction (30 mg / Kg) of PR8 + Oseltamivir (5 mg / Kg) + 30% EtOH extract 30 10 50 NBuOH fraction (30 mg / Kg) of PR8 + Oseltamivir (5 mg / Kg) + 30% EtOH extract 30 10 60 Water residue (30 mg / Kg) of PR8 + Oseltamivir (5 mg / Kg) + 30% EtOH extract 30 10 80 PR8 + 1-adamatylamine (10mg / Kg) 5 20 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Lobe 30% EtOH Extract (30mg / Kg) 30 5 20 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Leaf 70% EtOH Extract (30mg / Kg) 30 5 40 Hexane fraction of PR8 + 1-adamatylamine (10mg / Kg) + 30% EtOH extract 30 10 40 CH8 ₃ fraction (30 mg / Kg) of PR8 + 1-adamatylamine (10 mg / Kg) + 30% EtOH extract 30 9 50 NBuOH fraction (30mg / Kg) of PR8 + 1-adamatylamine (10mg / Kg) + 30% EtOH extract 30 10 50 Water residue (30mg / Kg) of PR8 + 1-adamatylamine (10mg / Kg) + 30% EtOH extract 30 10 60

1-4 Ivy  Isolated from extract Hederacoside  Antiviral Effects of C Compounds

Mice were infected with 10 ⁵ pfu of influenza, and mice were orally administered with Oseltamivir and hederacoside C, and the survival rate of the mice was confirmed. In the case of water fraction, it was confirmed that the treatment effect by Oseltamivir alone was increased significantly when co-administered with Oseltamivir, and it was confirmed that administration of hederacoside C was also active (Table 3).

Coadministration with Ivy Leaf Water Extract and Hederacoside C or Antiviral Agents Group Dose (mg / Kg) n 15 days after infection
Survival rate (%) PR8-treated - 9 0 Ivy Leaf Water Extract (100mg / Kg) 100 10 10 PR8 + Hederacoside C (10mg / Kg) 10 10 10 PR8 + Hederacodise C (50mg / Kg) 50 10 10 PR8 + Oseltamivir (5mg / Kg) 30 10 40 PR8 + Oseltamivir (5mg / Kg) + Ivy Leaf Water Extract (100mg / Kg) 100 5 50 PR8 + Oseltamivir (5mg / Kg) + Hederacoside C (10mg / Kg) 110 5 80 PR8 + Oseltamivir (5mg / Kg) + Hederacodise C (50mg / Kg) 50 5 80 PR8 + 1-adamatylamine (10mg / Kg) 5 40 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Leaf Water Extract (100mg / Kg) 100 5 60 PR8 + 1-adamatylamine (10mg / Kg) + Hederacoside C (10mg / Kg) 10 5 60 PR8 + 1-adamatylamine (10mg / Kg) + Hederacodise C (50mg / Kg) 50 5 60

Experimental Example 2. Toxicity Test

Acute toxicity was tested in ICR mice (male, 6 weeks, purchased from Aruet) according to KFDA specifications. As a result, 30% ivy leaf extract, 70% ivy leaf extract, chloroform fraction, and water residue did not show acute toxicity until oral administration of 2 g / kg.

As a result of the above experiments ivy leaf extracts and fractions can be safely used in influenza virus (infuenza virus) infectious diseases, especially when co-administered with antiviral agent was high activity.

The pharmaceutical composition containing the extract or the compound of the present invention will be described by way of example, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

HH30E 300 mg

Lactose 100 mg

10 mg talc

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

HH70E 300 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

HH-C 300 mg

3 mg of microcrystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

Hederacoside C Compound 300 mg

Di-mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

HH-W 300 mg

10 g of isomerized sugar

5 g of di-mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

Hederacoside C Compound 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin B ₁

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

HH30E 300 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated It is used to prepare a healthy beverage composition of the present invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (12)

Pharmaceutical composition for the prevention and treatment of infection by influenza virus containing ivy leaf extract or Hederacoside C isolated from it as an active ingredient. The method of claim 1,
The ivy leaf extract is a pharmaceutical composition, characterized in that crude extract, polar solvent soluble extract or non-polar solvent soluble extract. The method of claim 2,
The crude extract is a pharmaceutical composition, characterized in that the extract is soluble in a solvent selected from lower alcohols having 1 to 4 carbon atoms, such as water, methanol, ethanol, butanol, or a mixed solvent thereof. The method of claim 2,
The polar solvent soluble extract is a pharmaceutical composition, characterized in that the extract fraction soluble in the solvent selected from the remaining water, methanol, butanol or a mixed solvent thereof after removing the non-polar solvent soluble fractions from the crude extract. The method of claim 2,
The non-polar solvent soluble extract is a pharmaceutical composition, characterized in that the extract fraction purified only the extract soluble in hexane, methylene chloride, chloroform, or ethyl acetate solvent from the crude extract. A pharmaceutical composition for the prevention and treatment of infectious diseases caused by influenza virus, which contains a combination of ivy leaf extract or hederacoside C and an existing anti-influenza agent as an active ingredient. The method according to claim 6,
The existing anti-influenza agent is a pharmaceutical composition, characterized in that selected from Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel), Rimantadine, Rivaririn, or Taribavirin. 7. The method according to claim 1 or 6,
The influenza virus is H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 or H10N7. 7. The method according to claim 1 or 6,
The influenza virus-associated infection is a bird flu (bird flu), a pandemic flu, a pharmaceutical composition characterized in that the flu. Health functional food for the prevention and improvement of infectious diseases caused by influenza virus containing ivy leaf extract or Hederacoside C isolated from it as an active ingredient. Health functional food for the prevention and improvement of the infection caused by influenza virus containing a combination of ivy leaf extract, hederacoside C isolated from it and an existing antiviral agent as an active ingredient. The method according to claim 10 or 11
The health functional food is a health functional food in the form of a food, a powder, a granule, a tablet, a capsule, a syrup, a beverage, a gum, a tea, a vitamin complex, or a health functional food.

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