KR101543720B1 - A composition comprising the ivy leaf extract and the compound isolated therefrom for preventing and treating enterovirus-involved diseases - Google Patents

Abstract

The present invention relates to a composition containing an ivy leaf extract and a compound isolated therefrom as an active ingredient. More particularly, the present invention relates to an ivy leaf extract and a compound isolated therefrom, The composition is useful as a pharmaceutical composition or health functional food for preventing and treating infectious diseases caused by enteroviruses.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for preventing and treating an enterovirus-related disease containing an extract of Ivy leaf and a compound isolated therefrom as an active ingredient,

The present invention provides a composition for preventing and treating an enterovirus-related disease containing an extract of Ivy leaf and a compound isolated therefrom as an active ingredient.

[1] Racaniello VR, Baltimore D. 1981. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci US A. 78: 4887-91

[Literature 2] Racaniello VR, Baltimore D. 1981. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci US A. 78: 4887-91

[3] Chan YF, Sam IC, AbuBakar S. 2010. Phylogenetic designation of enterovirus 71 genotypes and subgenotypes using complete genome sequences. Infect Genet Evol . 10: 404-12;

[Document 4] Huang YP, Lin TL, Hsu LC, Chen YJ, Tseng YH, Hsu CC, Fan WB, Yang JY, Chang FY, Wu HS. 2010. Genetic diversity and C2-like subgenotype strains of enterovirus 71, Taiwan, 2008. Virol J. 20: 7: 277

[5] HyypiT, Horsnell C, Maaronen M, Khan M, Kalkkinen N, Auvinen P, Kinnunen L, Stanway G. 1992. A distinct picornavirus group identified by sequence analysis. Proc Natl Acad Sci US A. 89: 8847-51;

[Literature 6] Zell R, Stelzner A. 1997. Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5'- and 3'-nontranslated regions. Virus Res . 51: 213-29;

[Literature 7] Mayo MA, Pringle CR. 1998. Virus taxonomy - 1997. J Gen Virol . 79: 649-57;

[Document 8] Pringle CR. 1999. Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Arch Virol . 144: 2065-70

[9] Nasri D, Bouslama L, Pillet S, Bourlet T, Aouni M, Pozzetto B. 2007. Basic rationale, current methods and future directions for molecular typing of human enterovirus. Expert Rev Mol Diagn . 7: 419-34.

[Literature 10] Brown BA, Oberste MS, Alexander JP Jr., Kennett ML, Pallansch MA. 1999. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol . 73: 9969-75

[Document 11] Umakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G, Vasilenko S, Brodvarova I, Nikolova M, Gyurova S, Gacheva M, Mitov G, Ninov N, Tsylka E, Robinson I, Frolova M, Bashkirtsev V, Martiyanova L, Rodin V. 1979. Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol . 60: 329-40;

[Document 12] McMinn PC. 2002. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev. 26: 91-107;

[Literature 13] Wang SM, Lei HY, Huang KJ, Wu JM, Wang JR, Yu CK, Su IJ, Liu CC. 2003. Pathogenesis of enterovirus 71 Brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis . 188: 564-70

[Literature 14] Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, Bai R, Li X, Yan D, Wang H, Zhang Y, Zhu Z, Tan X, An H, Xue A Xu W. 2011. Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One . 6: e27895

[Document 15] Schmidt NJ, Lennette EH, Ho HH. 1974. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis . 129: 304-9

[16] Abubakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam SK. 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res . 61:19;

[Document 17] Shimizu H, Utama A, Yoshii K, Yoshida H, Yoneyama T, Sinniah M, Yusof Ma, Okuno Y, Okabe N, Shih SR, Chen HY, Wang GR, Kao CL, Chang KS, A. 1999. Enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in Malaysia, Japan and Taiwan in 1997-1998. Jpn J Infect Dis . 52: 125;

[Literature 18] Ho M. 2000. Enterovirus 71: the virus, its infections and outbreaks. J Microbiol Immunol Infect . 33: 20516;

[Literature 19] Singh S, Chow VT, Chan KP, Ling AE, Poh CL. 2000. RT-PCR, nucleotide, amino acid and phylogenetic analysis of enterovirus type 71 strains from Asia. J Virol Methods . 88: 193204;

[Literature 20] Kehle J, Roth B, Metzger C, Pfitzner A, Enders G. 2003. Molecular characterization of an Enterovirus 71 causing neurological disease in Germany. J Neurovirol . 9: 1268;

[Document 21] Mcminn P, Stratov I, Nagarajan L, Davis S. 2001b. Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis . 32: 23642;

[Literature 22] Yi L, Lu J, Kung HF, He ML. 2011. The virology and developments toward control of human enterovirus 71. Crit Rev Microbiol . 37: 313-27

[Literature 23] Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, Bai R, Li X, Yan D, Wang H, Zhang Y, Zhu Z, Tan X, An H, Xue A Xu W. 2011. Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One . 6: e27895.

[24] Cardosa MJ, Perera D, Brown BA, Cheon D, Chan HM, Chan KP, Cho H, McMinn P. 2003. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis the VP1 and VP4 genes. Emerg Infect Dis . 9: 461-468

[Document 25] Jassim SA, Naji MA. 2003. Novel antiviral agents: a medicinal plant perspective. J Appl Microbiol . 95: 412-27

[Document 26] Cowan MM. 1999. Plant products as antimicrobial agents. Clin Microbiol Rev. 12: 564-82;

[Document 27] Briskin DP. 2000. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol . 124: 507-14;

[Document 28] Williams JE. 2001. Review of antiviral and immunomodulating properties of the Peruvian rainforest with a particular emphasis on Una de Gato and Sangre de Grado. Altern Med Rev. 6: 567-79

[Literature 29] Venkateswaran PS, Millman I, Blumberg BS. 1987. Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies. Proc Natl Acad Sci US A. 84: 274-8;

[Literature 30] Thyagarajan SP, Subramanian S, Thirunalasundari T, Venkateswaran PS, Blumberg BS. 1988. Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus. Lancet . 2: 764-766

[Literature 31] Berg AK, Olsson A, Korsgren O, Frisk G. 2007. Antiviral treatment of Coxsackie B virus infection in human pancreatic islets. Antiviral Res . 74: 65-71

[Literature 32] Pevear DC, Tull TM, Seipel ME, Groarke JM. 1999. Activity of pleconaril against enteroviruses. Antimicrob Agents Chemother . 43: 2109-2115;

[Literature 33] Pevear DC, Hayden FG, Demenczuk TM, Barone LR, McKinlay MA, Collett MS. 2005. Relationship of pleconaril susceptibility and clinical outcomes in common colds caused by rhinoviruses. Antimicrob Agents Chemother . 49: 4492-4499;

[Document 34] Barnard DL. 2006. Current status of anti-picornavirus therapies. Curr Pharm Des . 12: 1379-90

[Literature 35] Graci JD, Cameron CE. 2006. Mechanisms of action of ribavirin against distinct viruses. Rev Med Virol . 16: 37-48

[Patent Document 36] Korean Patent Publication No. 10-2004-106201

[37] Demirci et al., Pharmazie 59, 770-774, 2004:

[Document 38] Korean Patent Laid-open Publication No. 10-2006-8310

[39] Majester-Savornin et al., Planta Med. 57, 260-262, 1991;

[Literature 40] Exp. Parasitol. 116, 340-345, 2007

[Literature 41] Mendel et al., J. Ethnopharmacol. 134, 796-802, 2011

[Document 42] Korean Patent Publication No. 10-2013-0081478

[Patent Document 43] Korean Patent Publication No. 10-2013-0081479

[Literature 44] Choi HJ, Lim CH, Song JH, Baek SH, Kwon DH. 2009. Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses. Phytomedicine . 16: 35-9;

[45] Choi HJ, Song JH, Park KS, Kwon DH.2009. Inhibitory effects of quercetin 3-rhamnoside on influenza virus replication. Eur J Pharm Sci . 37: 329-333.

Enteroviruses have a 27- to 30-nm envelope and are in the form of a regular dodecahedron with a single stranded positive sense RNA of approximately 7.2 to 7.5 kb in size (Figure 1). The ORF consists of a single open reading frame (ORF) and a non-coding region (NCR) that is not expressed at the 5'and 3'ends. The ORF occupies about 90% of the entire gene and is expressed as a polyprotein. It is composed of about 2,185 amino acids [Racaniello VR, Baltimore D. 1981. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci US A. 78: 4887-91.].

This polyprotein is divided into several different proteins by the proteolytic enzyme of the virus. One polyprotein is divided into P1, P2, and P3. P1 is the structural protein part, which encodes VP4, VP2, VP3 and VP1, which are the constituents of the virus's capsid protein. P2 and P3 parts are 2A, (Chan YF, Sam IC, AbuBakar S. 2010. Phylogenetic designation of enterovirus 71 genotypes and subgenotypes using complete genome sequences. Infect Genet Evol . 10: 404-12; Huang YP, Lin TL, Hsu LC, Chen YJ, Tseng YH, Hsu CC, Fan WB, Yang JY, Chang FY, Wu HS. 2010. Genetic diversity and C2-like subgenotype strains of enterovirus 71, Taiwan, 2008. Virol J. 20: 7: 277.].

Enteroviruses belong to the genus Picornaviridae, and about 68 serotypes have been known to date. There were three serotypes of poliovirus (PV: 1 ~ 3), 23 serotypes of Coxsackievirus A (CVA: 1 ~ 22, 24) and 6 serotypes of Coxsackievirus B (CVB: 6) and 28 serotypes of Echovirus (ECV: 1 to 7, 9, 11 to 21, 24 to 27, 29 to 33) and other human enteroviruses (EV: 68 to 116) , Horsnell C, Maaronen M, Khan M, Kalkkinen N, Auvinen P, Kinnunen L, Stanway G. 1992. A distinct picornavirus group identified by sequence analysis. Proc Natl Acad Sci US A. 89: 8847-51; Zell R, Stelzner A. 1997. Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5'- and 3'-nontranslated regions. Virus Res . 51: 213-29; 6. Mayo MA, Pringle CR. 1998. Virus taxonomy - 1997. J Gen Virol . 79: 649-57; 7. Pringle CR. 1999. Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Arch Virol . 144: 2065-70). However, in recent years, the genotypes have been classified into four species: HEV-A, HEV-B, HEV-C and HEV-D. Enterovirus 71 belongs to the HEV-A group [Nasri D, Bouslam L, Pillet S, Bourlet T, Aouni M, Pozzetto B. 2007. Basic rationale, current methods and future directions for molecular typing of human enterovirus. Expert Rev Mol Diagn . 7, pp. 419-34.] The subtypes of VP1 protein, a component of the capsid protein, are classified into subgenotypes such as A, B0-B5 and C1-C5 [Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, MA. 1999. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol . 73: 9969-75.].

It is known that enterovirus 71 is a major cause of neurological diseases such as encephalitis and poliomyelitis [Umakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G, Vasilenko S, Brodvarova I, Nikolova M, Gyurova S, Gacheva M, Mitov G, Ninov N, Tsylka E, Robinson I, Frolova M, Bashkirtsev V, Martiyanova L, Rodin V. 1979. Enterovirus 71 isolated from cases of epidemic poliomyelitis- like disease in Bulgaria. Arch Virol . 60: 329-40; McMinn PC. 2002. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev. 26: 91-107; Wang SM, Lei HY, Huang KJ, Wu JM, Wang JR, Yu CK, Su IJ, Liu CC. 2003. Pathogenesis of enterovirus 71 Brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis . 188: 564-70.]. The elderly and infants under five years of age are mainly caused by enterovirus type 71 and coxsakiovirus type A16. [Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, Bai R, Li X , Yan D, Wang H, Zhang Y, Zhu Z, Tan X, An H, Xu A, Xu W. 2011. Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One . 6: e27895.]. Although cockroach virus A16-type humoral disease usually presents with mild symptoms, enteric virus 71-type humoral disease can lead to neurological symptoms and death.

Enterovirus 71 was first detected in the United States in 1969 [Schmidt NJ, Lennette EH, Ho HH. 1974. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis . 129: 304-9.] In Malaysia, Singapore, Japan, Australia, USA, Germany, Thailand, China and many other countries. Large - scale outbreaks of enterovirus 71 caused by high mortality rates in recent years have occurred in East Asian countries. From 2008 to 2010, 299,000 people with enterovirus 71 had been developed in China and 1253 of them died. [Abubakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, and Lam SK. 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res . 61:19; 16. Shimizu H, Utama A, Yoshii K, Yoshida H, Yoneyama T, Sinniah M, Yusof Ma, Okuno Y, Okabe N, Shih SR, Chen HY, Wang GR, Kao CL, Chang KS, Miyamura T, Hagiwara A. 1999. Enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in Malaysia, Japan and Taiwan in 1997-1998. Jpn J Infect Dis . 52: 125; 17. Ho M. 2000. Enterovirus 71: the virus, its infections and outbreaks. J Microbiol Immunol Infect . 33: 20516; Singh S, Chow VT, Chan KP, Ling AE, Poh CL. 2000. RT-PCR, nucleotide, amino acid and phylogenetic analysis of enterovirus type 71 strains from Asia. J Virol Methods . 88: 193204; 19 Kehle J, Roth B, Metzger C, Pfitzner A, Enders G. 2003. Molecular characterization of an Enterovirus 71 causing neurological disease in Germany. J Neurovirol . 9: 1268; 20. Mcminn P, Stratov I, Nagarajan L, Davis S. 2001b. Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis . 32: 23642; 21. Yi L, Lu J, Kung HF, He ML. 2011. The virology and developments toward control of human enterovirus 71. Crit Rev Microbiol. 37: 313-27.]. During this period, the genital wart disease was C4a, a subgenotype of enterovirus type 71 [Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, Bai R, Li X, Yan D, Wang H, Zhang Y, Zhu Z, Tan X, An H, Xu A, Xu W. 2011. Emergence and transmission pathways of rapidly evolving branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One . 6: e27895.]. In Korea, enterococcal 71 C3 type was prevalent in 2000 [Cardosa MJ, Perera D, Brown BA, Cheon D, Chan HM, Chan KP, Cho H, McMinn P. 2003. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis . 9: 461-468.]. However, no vaccines or remedies were available during this period.

Due to the increase in viral resistance to currently developed antiviral agents, there have been recently developed antiviral agents having safety, higher efficacy, and lower production cost, but there are still no agents that have therapeutic effects in many viruses [Jassim SA, Naji MA. 2003. Novel antiviral agents: a medicinal plant perspective. J Appl Microbiol . 95: 412-27). For that reason, scientists in Asia, Europe and the United States are using data on antiviral agents from traditional medicinal plant-based medicines [Cowan MM. 1999. Plant products as antimicrobial agents. Clin Microbiol Rev. 12: 564-82; 26. Briskin DP. 2000. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol . 124: 507-14; 27. Williams JE. 2001. Review of antiviral and immunomodulating properties of the Peruvian rainforest with a particular emphasis on Una de Gato and Sangre de Grado. Altern Med Rev. 6: 567-79.]. Medicinal plants often exhibit several antiviral effects, which have low potential for side effects and tolerance and low production costs. Many medicinal plants have already been shown to have potent antiviral effects in viral infected individuals and animals [Venkateswaran PS, Millman I, Blumberg BS. 1987. Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies. Proc Natl Acad Sci US A. 84: 274-8; Thyagarajan SP, Subramanian S, Thirunalasundari T, Venkateswaran PS, Blumberg BS. 1988. Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus. Lancet . 2: 764-766.].

The symptoms of Enterovirus are generally not fatal, but Enterovirus 71 causes death and even leads to worms with neurological symptoms. Therefore, it is necessary to develop a therapeutic or preventive vaccine against Enterovirus 71.

Currently, the development of preventive vaccines and therapies for Enterovirus 71 is active, but there are no vaccines or therapeutic agents available yet. Pleconaril, developed as a remedy for the virus belonging to Picronaviridae, is known to bind to the capsid proteins of viruses and to inhibit the binding of susceptible cells to receptors [Berg AK, Olsson A, Korsgren O, Frisk G 2007. Antiviral treatment of Coxsackie B virus infection in human pancreatic islets.Antiviral Res . 74: 65-71] Clinically 78%, virologically 92%, and experimentally 88% have been reported to have improved patients, but were not approved by the FDA for adverse effects, Use is mostly limited to life-threatening situations, and resistant viruses have also been reported [Pevear DC, Tull TM, Seipel ME, Groarke JM. 1999. Activity of pleconaril against enteroviruses.Antimicrob Agents Chemother . 43: 2109-2115; Pevear DC, Hayden FG, Demenczuk TM, Barone LR, McKinlay MA, Collett MS. 2005. Relationship of pleconaril susceptibility and clinical outcomes in common colds caused by rhinoviruses.Antimicrob Agents Chemother . 49: 4492-4499; Barnard DL. 2006. Current status of anti-picornavirus therapies.Curr Pharm Des . 12: 1379-90.]. Ribavirin, an antiviral agent showing antiviral activity against DNA viruses as well as RNA viruses, has also been reported to exhibit drug resistance in various viruses [Graci JD, Cameron CE. 2006. Mechanisms of action of ribavirin against distinct viruses.Rev Med Virol. 16: 37-48.]. Therefore, it is urgent to develop a broad-spectrum therapeutic agent for various subgenotypes of enterovirus 71 without side effects.

Meanwhile, Hedera helix ) is a plant called English ivy. Leaf extract is used for the treatment of bronchitis due to its genetic effect (Korean Patent Laid-Open No. 10-2004-106201), and its main component is saponin components called hederacoside Demirci et al., Pharmazie 59, 770-774, 2004: Korean Patent Publication 10-2006-8310). In addition to their genetic effects, the extracts of these leaves have been reported to have antibacterial, antifungal, antiresorance, and digestive action (Majester-Savornin et al., Planta Med. 57, 260-262, 1991; Exp. In addition, the inventors of the present invention have found that the use of ivy extract and the compounds isolated therefrom as an agent for the treatment of infectious diseases caused by influenza viruses (Korean Patent Application Laid- Japanese Patent Application Laid-Open No. 10-2013-0081478), and an invention for use as an agent for the treatment of infectious diseases caused by influenza virus in combination with ivy and thymus extract (Korean Patent Laid-Open No. 10-2013-0081479).

However, none of the above references discloses or discloses the inhibitory activity of ivy leaf extracts and compounds isolated therefrom against enteroviruses.

Therefore, the present inventors have conducted experiments on the inhibition of cellular lesions by Enterobirus 71 subgenotype C3 and C4a using Vero cells for ivy leaf extract and hederasaponin B isolated therefrom for use as a therapeutic agent for enterovirus infection The present inventors completed the present invention by confirming that the samples of the present invention showed a strong inhibitory effect on Enteroprovirus 71 subgenotype C3 and C4a-induced cell lesions in the experiment for inhibiting viral VP protein expression.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of infectious diseases caused by enterovirus containing ivory leaf extract or hederasaponin B isolated therefrom as an active ingredient.

The present invention also provides a health functional food for preventing and ameliorating infectious diseases caused by enterovirus containing ivory leaf extract or hederasaponin B isolated therefrom as an active ingredient.

The ivy leaf extract as defined herein is characterized by being crude extract, polar solvent-soluble extract or nonpolar solvent-soluble extract.

The crude extract as defined in the present application may be dissolved in a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol and butanol, or a mixed solvent thereof, preferably water and ethanol mixed solvent, (V / v) ethanol, more preferably 10 to 50% (v / v) ethanol.

The non-polar solvent soluble extract fractions defined herein comprise extract fractions which are soluble in non-polar solvents which have been purified from the crude extracts of the present invention and purified only in extracts soluble in hexane, methylene chloride, chloroform or ethyl acetate, preferably ethyl acetate solvent.

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.

The present invention also provides a pharmaceutical composition for the prevention and treatment of infectious diseases caused by enterovirus comprising, as an active ingredient, a combination of ivy leaf extract or hederasaponin B isolated from the extract and an existing anti-enteroviral agent.

The present invention also provides a health functional food for prevention and improvement of infectious diseases caused by enterovirus, which contains, as an active ingredient, a combination of ivory leaf extract or hederasaponin B isolated from the extract and an existing anti-enteroviral agent do.

The existing anti-enteroviral agent as defined herein is Pleconaril.

The enteroviruses as defined herein are characterized by having serotypes of HEV-A, HEV-B, HEV-C or HEV-D, preferably HEV-A serotypes, more preferably Enterovirus 71 serotypes .

Infectious diseases caused by enteroviruses as defined herein include, but are not limited to, conjunctivitis, otitis media, aspergillosis, aseptic meningitis, herpes zoster, wrist disease, testisitis, myocarditis, encephalitis, Guillian-Barre syndrome, ataxia, , Transverse myelitis, limb paralysis, diabetes, epidemic hemorrhagic keratoconjunctivitis, and the like.

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.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid Citric acid, lactic acid, glycollic acid, gluconic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid can be used.

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.

The ivy leaf extract of the present invention can be prepared as follows. Dried ivy leaves are washed with water and then washed with water and then washed with water, a solvent selected from lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, and butanol or a mixed solvent thereof, preferably water and ethanol mixed solvent, After 10 to 100% ethanol is mixed several times, the mixture is subjected to ultrasonic extraction, hot water extraction and room temperature extraction at 30 to 150 ° C, preferably 50 to 100 ° C for 30 minutes to 48 hours, preferably 1 to 12 hours Or a reflux extraction method, preferably an ultrasonic extraction method, is repeatedly performed about 1 to 20 times, preferably 2 to 10 times, and the resulting extract is filtered, concentrated under reduced pressure, and dried to obtain a crude extract of the present invention.

The polar solvent or the non-polar solvent soluble extract of the present invention may contain about 0.0005 to 0.005 times, preferably 0.05 to 0.5 times the volume (v / w%) of the crude extract, preferably 10 to 90% ), Followed by fractionation using ethyl acetate and butanol to obtain a nonpolar solvent-soluble fraction extracted with n-hexane, methylene chloride, ethyl acetate and the like; And polar solvent-soluble extract fractions soluble in polar solvents such as butanol and water can be obtained.

The non-polar solvent-soluble fraction extracted in the nonpolar solvent, such as n-hexane, methylene chloride or ethyl acetate, obtained in the above, is dissolved in hexane and methylene chloride mixed solvent in a flash column The purification method using chromatography such as chromatography, RP C18 column chromatography or silica gel open column chromatography or Diaion HP-20 column chromatography is repeated several times to obtain the compound of the present invention, hederasaponin B, Can be purified and obtained, respectively.

The inventors of the present invention found that the extracts and compounds obtained by the above-described method were used for the inhibition of cell-lesion inhibition by enterovirus 71 subgenotype C3 and C4a using Vero cells and the inhibition of viral VP protein expression by Enterobirus 71 subgenotype C3 And C4a, it was confirmed that the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of infectious diseases caused by enteroviruses.

Accordingly, the present invention provides a pharmaceutical composition for prevention and treatment of infectious diseases caused by enterovirus comprising the extract of Ivy leaf obtained by the above production method, and a compound isolated therefrom as an active ingredient.

In addition, the ivy leaf has been used for a long time as a herbal medicine, and the compound isolated from the ivy leaf extract of the present invention has no 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.

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The 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 pharmacologically active compounds as well as in suitable aggregates.

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 sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol and vegetable oils such as olive oil, and injectable esters such as ethyl oleate. 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 the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, once or several times per day. In the composition, the compound of the present invention may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the entire 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 may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine and intracerebroventricular injections.

The present invention also provides a health functional food for preventing and ameliorating infectious diseases caused by enterovirus comprising the extract of Ivy leaf obtained by the above production method or a compound isolated therefrom as an active ingredient.

The extract or the compound of the present invention can be used variously for medicines, foods and beverages for the prevention and treatment of the objective disease. Examples of the food to which the extract or the compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes and health supplements. Examples of the foods include powders, granules, tablets, 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 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 0.02 to 30 g, Preferably in a proportion of 0.3 to 10 g.

The health beverage composition of the present invention contains the above extract or compound as an essential ingredient in the indicated ratio, and there is no particular limitation on the liquid ingredient, and it may contain various flavors or natural carbohydrates as an additional ingredient have. 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. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 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 may 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 described above, the ivy leaf extract of the present invention or the compound isolated therefrom can be used for inhibiting the cell-lesion-suppressing effect of enterovirus 71 subgenotype C3 and C4a using Vero cells and inhibiting the expression of viral VP protein In the experiment, by confirming that the samples of the present invention exhibit a strong inhibitory effect on cell lesions caused by enterovirus 71 subgenotype C3 and C4a, the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of infectious diseases caused by enterovirus .

Figure 1 shows photographs showing the effect of Enterovirus 71 (EV71) C3 on cytopathic effect (A) non-infected cells, (B) non-infected cells treated with hederasaponin B, (C) ribavirin (E) EV71 C3-infected cells treated with (E) hederasaponin B; (F) EV71 C3- infected cells treated with ribavirin;
Figure 2 shows photographs showing the effect of Enterovirus 71 (EV71) C4a on cytopathic effect (A) non-infected cells, (B) non-infected cells treated with hederasaponin B, (C) ribavirin (D) EV71 C4a-infected cells, (E) EV71 C4a-infected cells treated with hederasaponin B, (F) EV71 C4a-infected cells treated with ribavirin;
FIG. 3 shows the inhibitory effect of the samples on VP1 expression. FIG.

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 the test of Professor Kwon Yong-Soo (Kangwon National University, College of Pharmacy, Kangwon National University, yskwon@kangwon.ac.kr) were added to various concentrations of water and ethanol mixed solvent, ie, 30% v / v) ethanol solvent was added and the mixture was extracted three times with an ultrasonic wave extractor (8510 DHT; CT, USA) for 3 hours at 60 ° C, followed by vacuum filtration to recover the supernatant. This procedure was repeated four times, and the supernatant was collected and dried with a reduced pressure condenser and freeze dryer (FD5512, Ilshin, Seoul, Korea) to obtain 500 g of a 30% (v / v) ethanol mixed solvent extract of ivy leaves (Hereinafter referred to as " HH30E ").

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

The dried 30% ethanol extract (500 g) obtained in Example 1-1 was suspended in distilled water and fractionated with ethyl acetate (ethylacetate) and n-butanol (BuOH) according to a fractionation method common in the art And dried under reduced pressure to obtain 23 g of the ethyl acetate-soluble fraction and 150 g of the butanol-soluble fraction, and lyophilized (hereinafter referred to as HH-EA and HH-BU, respectively).

Example   2. Ivy leaves  Isolation of the active compound from the extract

In order to identify the active substance, 100 g of the butanol-soluble fraction of the highest activity fraction was fractionated by Diaion HP-20 column chromatography using water: methanol 100: 0, 80:20, 60:40, 40:60 , 20:80, and 0: 100 in a mixed solvent. Six fractions were separated by a concentration gradient method. Using the 30% ethanol extract of Hedera helix and 6 fractions, antiviral activity was confirmed in C3 and C4a subtypes of enterovirus71 (Table 1). The active fractions exhibiting antiviral activity were concentrated and the active fractions were dissolved again in methanol, and the active material was separated by high performance liquid chromatography (HPLC) at a concentration of water: methanol (30:70). ESI-MS, ^{As a result of 1} H NMR and ¹³ C NMR analysis, the structure of hederasaponin B (Fig. 1) was compared with that of existing literature data.

EV71 C3  And C4a For Ivy  The extract and Fraction  Antiviral activity. EV71 C3 EV71 C4a Materials tested CC ₅₀ ^a IC ₅₀ ^b TI ^c CC ₅₀ ^a IC ₅₀ ^b TI ^c Hedera Helix 30% EtOH extract > 50 6.58 ± 0.11 7.59 > 50 22.00 ± 2.06 4.55 100% H ₂ O fraction > 50 ND ^d - > 50 ND ^d - 20% MeOH fraction > 50 25.23 + - 4.93 1.98 > 50 ND ^d - 40% MeOH fraction > 50 7.92 ± 1.44 6.31 > 50 43.12 ± 1.91 1.16 60% MeOH fraction > 50 2.75 ± 1.05 18.18 > 50 47.10 ± 5.16 1.06 80% MeOH fraction > 50 38.22 + - 4.13 1.31 > 50 ND ^d - 100% MeOH fraction 31.03 ND ^d - 29.76 ND ^d - Results are presented as the mean IC ₅₀ values obtained from three independent experiments carried out in triplicate ± SD
^a Concentration required to reduce cell growth by 50% (/ / ml).
^b Concentration required to inhibit virus-induced CPE by 50% (㎍ / ml).
^c Therapeutic index = CC ₅₀ / IC ₅₀
^d Not determined

Reference Example 1. Viruses, cell lines and reagents

Enterovirus was obtained from the Vaccine Research Institute of the National Institute of Health (NIH) and propagated in vero cells (CCL-81, ATCC) at 37 ℃. Vero cells were maintained in MEM media (11095-080, Gibco) supplemented with 1% FBS (16000-044, Gibco) and 0.01% antibiotic (15070-063, Gibco). Antibiotics, trypsin EDTA, FBS and MEM media were purchased from Gibco. Tissue culture plates were purchased from Falcon, sulforhodamine B (SRB) was purchased from SigmaAldrich (S9012-25G), Ribavirin was purchased from DUCHEFA (R0182, Netherlands) and DMSO was used for dissolution of antiviral compounds. MEM media Lt; / RTI > The final concentration of DMSO was used as the maximum concentration of 0.1%, which had no effect on cell culture, and 0.1% DMSO was used as a cell control.

Experimental Example 1. Inhibitory activity against enterovirus

The antiviral activity and cytotoxicity of the samples obtained in the above Examples were assayed by SRB assay using the method described in the literature as follows [Choi HJ , Lim CH, Song JH, Baek SH, Kwon DH. 2009. Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses. Phytomedicine. 16: 35-9; Choi HJ, Song JH, Park KS, Kwon DH.2009. Inhibitory effects of quercetin 3-rhamnoside on influenza virus replication. Eur J Pharm Sci . 37: 329-333).

1-1. Antivirus  activation

Cells susceptible to each virus were prepared in each well of a 96-well culture plate (Nunc, Denmark) in an amount of 2 × 10 ⁴ . After 24 h, each cell was grown in a 96-well culture plate at 90%. Media was removed from each well and each cell was washed once with PBS. Then, 10 μL of hederasaponin B at a concentration appropriate for 90 μL of MEM containing 1% FBS and each virus was added. The concentration of Hederasaponin B was prepared by diluting 10 times. The antiviral activity of Hederasaponin B was determined in four concentration ranges from 0.4 μg / mL to 50 μg / mL. Four wells were used as the cell control without virus treatment and the other four wells were used as the cell control without virus and compound treatment. A CO ₂ incubator (VS-9108MS, Vision, Korea ) a phase contrast microscope in the form of after incubation for 2 days by 37 ℃, conditions of 5% CO ₂ cells; in (Axiovert 10 Zeiss, Wetzlar, Germany ) with 0.4 X 10 magnification After observation, images were recorded.

1-2. Cytotoxicity test

Each of the cells cultured on 96-well plates was treated with four concentrations of Hederasaponin B at a concentration of 0.4 μg / mL to 50 μg / mL, followed by incubation in a CO ₂ incubator at 37 ° C. for 2 days, The cytotoxicity was assessed by SRB assay.

1-3. Western blot analysis

To investigate the antiviral activity of hederasaponin B against enterovirus 71 subgenotype C3 and C4a, Western blot analysis was used for the VP protein of enterovirus 71.

Vero cells were cultured in 6-well culture plates at 5 × 10 ⁵ cells per well. After 24 hours, the culture solution was removed and washed with PBS. Then, hederasaponin B and ribavirin (100 μL) at a concentration of 50 μg / mL were added to 900 μL of virus culture medium containing 1% FBS. After incubation at 37 ° C in a 5% CO ₂ incubator for 48 hours, cells were lysed using RIPA lysis buffer (89900, Pierce). The dissolved cells were centrifuged to separate only the supernatant containing the protein. The protein-containing supernatant was heated at 100 ° C. for 10 minutes and then electrophoresed at 100 V for 1 hour using 12% acrylamide gel (456-1043, BIO-RAD). The protein in acrylamide gel was then transferred to iBlotTransfer Stack, PVDF, and regular-size (IB401001, Invitrogen) at 20V for 7 min using a device (iBlotGel Transfer Device, Invitrogen, Carlsbad, Calif.). The membranes were incubated in 5% skim milk (232100, Difco) for 1 hour at room temperature and washed 3 times with 1X PBST (Phosphate buffer saline Tween-20). The primary antibody diluted 1: 1000 in 5% skim milk was reacted with the mouse anti-enterovirus 71 monoclonal antibody (MAB 979, Millipore) for 2 hours at room temperature and then washed three times with PBST gave. In this experiment, α-tubulin, which was used as a control, was subjected to the same method using mouse monoclonal IgG1 (SC-32293, Santa Cruz) as a primary antibody. Enterovirus 71 C3 and C4a VP proteins and a secondary antibody to identify α-Tubulin as a control were polyclonal goat anti-mouse IgG (H + L) HRP (SA001-500, GenDEPOT). polyclonal goat anti-mouse IgG (H + L) HRP was diluted in 5% skim milk at a ratio of 1: 5000 and reacted at room temperature for 1 hour. Then, the cells were washed 3 times with PBST, Imaging System, BIO-RAD, Hercules, CA, USA).

1-4. Experiment result

1. Antiviral activity and cytotoxicity of hederasaponin B against enterovirus 71 subgenotype C3 and C4a

The cytotoxicity of Hederasaponin B and the antiviral effect of Enterovirus 71 subgenotype C3 and C4a were measured. Cytotoxicity of hederasaponin B on Vero cells used was not observed at the maximum concentration of 50 μg / mL. The IC ₅₀ values of Hederasaponin B, which showed 50% inhibition of viral activity, were 24.77 μg / mL for Enterovirus 71 C3 and 41.77 μg / mL for Enterovirus 71 C4a (Table 2). However, ribavirin, used as a positive control, did not show antiviral activity in Enterobirus 71 subgenotype C3 and C4a. Therefore, it was confirmed that hederasaponin B has a strong inhibitory effect on enteropirus 71 subgenotype C3 and C4a-induced cell lesions.

EV71 C3  And C4a For Hyderazaponin  Antiviral activity of B. EV71 C3 EV71 C4a Compound CC ₅₀ ^a IC ₅₀ ^b TI ^c CC ₅₀ ^a IC ₅₀ ^b TI ^c Hederasaponin B > 50 24.77 ± 12.56 2.02 > 50 41.77 + - 0.76 1.18 Ribavirin > 50 ND ^d - > 50 ND ^d - Results are presented as the mean IC ₅₀ values obtained from three independent experiments carried out in triplicate ± SD
^a Concentration required to reduce cell growth by 50% (/ / ml).
^b Concentration required to inhibit virus-induced CPE by 50% (㎍ / ml).
^c Therapeutic index = CC ₅₀ / IC ₅₀
^d Not determined

2. hederasaponin  Of B Enterovirus  71 subgenotype C3 Wow C4a On by Cell lesion  Inhibitory effect

Vero cells were infected with Enterovirus 71 subgenotype C3 and C4a. After 2 days, cell lesions were imaged using a microscope. (Fig. 2A), which had not been infected with viruses, and treated with 50 μg / mL of hederasaponin B (Fig. 1B) (Fig. 2B) without virus infection and 50 μg / mL of ribavirin In one group (Fig. 1C) (Fig. 2C), normal cells showed the same shape as normal controls. As a result, hederasaponin B and ribavirin did not show cytotoxicity. (Fig. 1D) infected with enterovirus 71 subgenotype C3 and C4a and treated with 50 μg / mL of hederasaponin B (Fig. 1D) infected with enterovirus 71 subgenotype C3 and C4a ) And ribavirin treated group (FIG. 1F) treated with 50 μg / mL of ribavirin (FIG. 2F) showed cell lesions due to the respective viruses in the control and ribavirin treated groups. In the hederasaponin B treated group, The lesion was suppressed and showed normal cell shape.

3. Hederasaponin  B virus VP  Inhibitory Effect on Protein Expression

Antiviral activity of Hederasaponin B against Enterovirus 71 subgenotype C3 and C4a was confirmed by Western blot analysis. Enterovirus 71 The VP protein of Enterovirus 71 was identified 48 hours after infection. Hederasaponin B inhibited the expression of each VP protein in Enterobirus 71 subgenotype C3 and 48 hours after C4a infection. However, ribavirin used as a control agent did not inhibit the expression of each viral VP protein at all (Fig. 3).

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% ivory leaf extract, 70% ivory leaf extract, ethyl acetate fraction, butanol fraction and hyderazaponin B showed no acute toxicity until oral administration of 2 g / kg.

As a result of the above experiment, ivy leaf extract, fraction and hyderazaponin B can be safely used for enterovirus infectious diseases, especially when they are co-administered with an antiviral agent.

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 hydrate --------------------------------------------- 100 mg

Talk ------------------------------------------------- --- 10 mg

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

Formulation example  2. Preparation of tablets

HH-EA ----------------------------------------------- --- 300 mg

Corn starch --------------------------------------------- 100 mg

Lactose hydrate --------------------------------------------- 100 mg

Magnesium stearate -------------------------------------- 2 mg

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-BU ----------------------------------------------- --- 300 mg

Microcrystalline cellulose ----------------------------------------- 3 mg

Lactose hydrate -------------------------------------------- 14.8 mg

Magnesium stearate ------------------------------------ 0.2 mg

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 injections

Hadera saponin B compound - 300 mg

Di-mannitol ---------------------------------------------- 180 mg

Sterile 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-BU ----------------------------------------------- --- 300 mg

Isseong Party ------------------------------------------------ - 10 g

Di-mannitol ----------------------------------------------- - 5 g

Purified water ------------------------------------------------- - Suitable amount

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

Hadera saponin B compound - 1000 mg

Vitamin mixture --------------------------------------------

Vitamin A Acetate ------------------------------------- 70 [mu] g

Vitamin E ----------------------------------------------- 1.0 Mg

Vitamin B ₁ ---------------------------------------------- 0.13 mg

Vitamin B ₂ ----------------------------------------------- 0.15 mg

Vitamin B ₆ ----------------------------------------------- - 0.5 mg

Vitamin B ₁₂ ----------------------------------------------- 0.2 [mu] g

Vitamin C ----------------------------------------------- 10 Mg

Biotin ------------------------------------------------- 10 [mu] g

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------------- - 50 [mu] g

Calcium pantothenate ----------------------------------------- 0.5 mg

Inorganic mixture -------------------------------------------

Ferrous sulfate 1.75 mg < RTI ID = 0.0 >

Zinc oxide - 0.82 mg

Magnesium carbonate ----------------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary 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

HH-EA ----------------------------------------------- - 300 mg

Vitamin C ------------------------------------------------ 15 g

Vitamin E (powder) ----------------------------------------- 100 g

Lactic Acid Iron ----------------------------------------------- 19.75 g

Zinc oxide ----------------------------------------------- 3.5 g

Nicotinic acid amide 3.5 g

Vitamin A ----------------------------------------------- 0.2 g

Vitamin B ₁ --------------------------------------------- 0.25 g

Vitamin B ₂ ---------------------------------------------- 0.3 g

Water ------------------------------------------------- ----- Quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The solution was filtered to obtain a sterilized 2-liter container, which was sealed and refrigerated It is used in the production of the health 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 (14)

A pharmaceutical composition for preventing and treating infectious diseases caused by enterovirus containing ivory leaf extract as an active ingredient. The method according to claim 1,
Wherein the ivy leaf extract is a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract. 3. The method of claim 2,
Wherein the crude extract is an extract soluble in a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof. 3. The method of claim 2,
Wherein the polar solvent-soluble extract is an extract fraction obtained by removing the non-polar solvent-soluble fractions from the crude extract and using the remaining fraction in a solvent selected from water, methanol, butanol or a mixed solvent thereof. 3. The method of claim 2,
Wherein the nonpolar solvent soluble extract is an extract fraction obtained by purifying only the extract soluble in hexane, methylene chloride, chloroform, or ethyl acetate solvent from the crude extract. delete delete The method according to claim 1,
Wherein said enterovirus is a serotype of HEV-A, HEV-B, HEV-C, or HEV-D. The method according to claim 1,
The infectious disease caused by the enterovirus may include at least one of conjunctivitis, otitis media, skin rash, aseptic meningitis, herpetic sinusitis, hydrocephalus, testisitis, myocarditis, encephalitis, Guillian-Barre syndrome, ataxia, Myelodysplasia, diabetic mellitus, or epidemic hemorrhagic keratoconjunctivitis. A health functional food for prevention and improvement of infectious diseases caused by enterovirus containing ivory leaf extract as an active ingredient. delete The method of claim 10, wherein
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. A pharmaceutical composition for preventing and treating infectious diseases caused by enterovirus containing hederasaponin B (Idera saponin B) isolated from ivy leaf extract as an active ingredient. A health functional food for preventing and improving infectious diseases caused by enterovirus containing hederasaponin B (Idera saponin B) isolated from ivy leaf extract as an active ingredient.

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