KR20150100306A - A composition comprising the Phyllanthus urinaria extract and the compound isolated therefrom for preventing and treating enterovirus-or Coxsackievirus involved diseases - Google Patents

Abstract

The present invention relates to a composition comprising a Phyllanthus urinaria extract and a compound isolated therefrom as an active ingredient and, specifically, samples of the present invention exhibits a strong inhibitory effect on CVA16 and EV71-induced cell lesions and confirms a strong anti-virus activity against CVA16 and EV71 virus-infected cells in experiments on CVA16 and EV71-induced cell lesions inhibitory effect and cytotoxicity targeting the extracts and the compounds using a Vero cell line. The composition is useful as a pharmaceutical composition or a health functional food for preventing and treating infections by enterovirus or Coxsackie virus.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for preventing and treating enterovirus or coxsackie virus-related diseases containing, as an active ingredient, a fox bead extract and a compound isolated therefrom, and a composition for preventing and treating Coxsackievirus related diseases}

The present invention provides a composition for the prevention and treatment of enterovirus or coxsackie virus-related diseases containing an extract of fox beads 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] 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;

[3] 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 4] 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

[5] 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 6] 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.

[7] 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 8] 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 9] 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 10] 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 11] Jassim SA, Naji MA. 2003. Novel antiviral agents: a medicinal plant perspective. J Appl Microbiol . 95: 412-27

[Document 12] 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;

[Document 13] 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 14] 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 15] 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 16] 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

[Reference 17] Pevear DC, Tull TM, Seipel ME, Groarke JM. 1999. Activity of pleconaril against enteroviruses. Antimicrob Agents Chemother . 43: 2109-2115;

[Literature 18] 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;

[Literature 19] Barnard DL. 2006. Current status of anti-picornavirus therapies. Curr Pharm Des . 12: 1379-90.

[Literature 20] Graci JD, Cameron CE. 2006. Mechanisms of action of ribavirin against distinct viruses. Rev Med Virol . 16: 37-48

(2): (1) (2): (1) (2): (1): (a)

Inhibition of enterovirus 71 (eV-71) infections by a novel antiviral peptide derived from eV-71 capsid protein VP1.PloS one (2012) 7 5): e34589.

[Literature 23] Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH: Virology, epidemiology, pathogenesis, and control of enterovirus 71. The Lancet infectious diseases (2010) 10 (11): 778-790.

[Document 24] Cheng A, Fung CP, Liu CC, Lin YT, Tsai HY, Chang SC, Chou AH, Chang JY, Jiang RH, Hsieh YC, meat al: A phase i, randomized, open-label study to evaluate the safety and immunogenicity of an enterovirus 71 vaccine. Vaccine (2013) 31 (20): 2471-2476.

[Literature 25] Shang L, Xu M, Yin Z: Antiviral drug discovery for enterovirus 71 infections. Antiviral research (2013) 97 (2): 183-194.

[Literature 26] Information Sourcing, Illustrated Essay, p766-767, Younglim, 1998

[27] Yuandani, Ilangkovan M, Jantan I, Mohamad HF, Husain K, Abdul Razak AF: Inhibitory effects of standardized extracts of phyllanthus amarus and phyllanthus urinaria and their marker compounds on phagocytic activity of human neutrophils. 2013) 2013, pp.603-634

[28] Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK: Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology: A review. Journal of ethnopharmacology (2011) 138 (2): pp.286-3134

[29] Zhao L, Zhang SL, Tao JY, Pang R, Jin F, Guo YJ, Dong JH, Ye P, Zhao HY, Zheng GH: Preliminary exploration of anti-inflammatory mechanism of corilagin (beta-1- galloyl-3,6- (r) -hexahydroxydiphenoyl-d-glucose) in vitro. International immunopharmacology (2008) 8 (7): 1059-1064;

[Document 30] Duan W, Yu Y, Zhang L: Antiatherogenic effects of phyllanthus emblica associated with corilagin and its analogue. Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan (2005) 125 (7): 587-591

[31] Duan W, Yu Y, Zhang L: Antiatherogenic effects of phyllanthus emblica associated with corilagin and its analogue. Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan (2005) 125 (7): 587-591;

[32] Moreira J, Klein-Junior LC, Cechinel Filho V, de Campos Buzzi F: Anti-hyperalgesic activity of corilagin, a tannin isolated from phyllanthus niruri l. (euphorbiaceae). Journal of Ethnopharmacology (2013) 146 (1): 318-323;

[Literature 33] Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK: Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology: A review. Journal of ethnopharmacology (2011) 138 (2): 286-313;

[Literature 34] Liu KC, Lin MT, Lee SS, Chiou JF, Ren S, Lien EJ: Antiviral tannins from two phyllanthus species. Planta Med (1999) 65 (1): 43-46;

[35] Jikai L, Yue H, Henkel T, Weber K: One step purification of corilagin and ellagic acid from phyllanthus urinaria using high-speed counter current chromatography. Phytochem Anal (2002) 13 (1): 1-3) .

[36] Kinoshita S, Inoue Y, Nakama S, Ichiba T, Aniya Y: Antioxidant and hepatoprotective actions of medicinal herb, terminal catapta l. From Okinawa island and its tannin coralagin. Phytomedicine : international journal of phytotherapy and phytopharmacology (2007) 14 (11): 755-762;

[Literature 37] Li Y, Yu S, Liu D, Proksch P, Lin W: Inhibitory effects of polyphenols toward hcv from the mangrove plant excoecaria agallocha l . Bioorganic & medicinal chemistry letters (2012) 22 (2): 1099-1102;

[Literature 38] Satomi H, Umemura K, Ueno A, Hatano T, Okuda T, Noro T: Carbonic anhydrase inhibitors from the pericarps of punica granatum l . Biological & pharmaceutical bulletin (1993) 16 (8): 787-790;

[39] Thitilertdecha N, Teerawutgulrag A, Kilburn JD, Rakariyatham N: Identification of major phenolic compounds from nephelium lappaceum l. And their antioxidant activities. Molecules ( Basel , Switzerland ) (2010) 15 (3): 1453-1465

[40] Park JH, Joo HS, Yoo KY, Shin BN, Kim IH, Lee CH, Choi JH, Byun K, Lee B, Lim SS, Kim MJ meat al : Extract from terminalia chebula seeds protect against experimental ischemic neuronal damage via maintaining sods and bdnf levels. Neurochemical research (2011) 36 (11): 2043-2050

[Literature 41] Hau DK, Zhu GY, Leung AK, Wong RS, Cheng GY, Lai PB, Tong SW, Lau FY, Chan KW, Wong W Y, Lam KH meat al : In vivo anti - tumor activity of corilagin on Hep3B hepatocellular carcinoma. Phytomedicine : international journal of phytotherapy and phytopharmacology (2010) 18 (1): 11-15

[Document 42] 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.

[43] Choi HJ, Song JH, Park KS, Kwon DH: Inhibitory effects of quercetin 3-rhamnoside on influenza virus replication. Eur J Pharm Sci (2009) 37 (3-4): 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.].

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.

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.].

Although the symptoms of Enterovirus are generally not fatal, Enterovirus 71 can cause death and even lead 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 has been actively conducted, 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.

Human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) virus are known to cause hand and foot and mouth disease (HFMD) in infants and adolescents [Mao Q, Wang Y, Yao X, Bian L, Wu Inhibition of Enterovirus 71 (2): Tan CW, Chan YF, Sim KM, Tan EL, Poh CL: Inhibition of Enterovirus 71 (X), Xu M, and Liang Z: Coxsackievirus a16: Epidemiology, ev-71) infections by a novel antiviral peptide derived from eV-71 capsid protein vp1.PloS one (2012) 7 (5): e34589.

Recently, there have been CVA16 and EV71 viruses in Western Asia, including Korea, Japan and China. In 2008, there has been an outbreak of foot and mouth disease. The hand-foot disease caused by CVA16 and EV71 is usually cured with mild symptoms, including blisters or ulcers on the hands, feet and lips, but occasionally, aseptic meningitis, encephalitis, It can cause serious complications on the central nervous system (CNS) such as myocarditis. [Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH: Virology, epidemiology, pathogenesis, and control of enterovirus 71. The Lancet infectious diseases (2010) 10 (11): 778-790.

However, a safe and effective vaccine against CVA16 and EV71 infection has not been developed, and appropriate therapeutic agents have not been developed [Cheng A, Fung CP, Liu CC, Lin YT, Tsai HY, Chang SC, Chou AH, Chang JY, Jiang RH, Hsieh YC, Su IJ meat al: A phase i, randomized, open-label study to evaluate the safety and immunogenicity of an enterovirus 71 vaccine. Vaccine (2013) 31 (20): 2471-2476., 25Shang L, Xu M, Yin Z: Antiviral drug discovery for enterovirus 71 infections. Antiviral research (2013) 97 (2): 183-194.

Importantly, in a recent study, coinfection of the CVA16 and EV71 viruses is known to cause serious neurological complications on the CNS and increase the possibility of genetic recombination, leading to HFMD outbreaks [Mao Q, Wang Y, Yao X, Bian L, Wu X, Xu M, Liang Z: Coxsackievirus a16: Epidemiology, diagnosis, and vaccine. Human vaccines & immunotherapeutics (2013) 10 (2).

Therefore, it is important to develop an antiviral agent that inhibits both CVA16 and EV71, such as fox bead extract of the present invention and collillazin, and is useful as a therapeutic agent for HFMD disease.

Meanwhile, the fox beads ( Phyllanthus urinaria ) is a perennial herb that belongs to the Euphorbiaceae. It is distributed in Jeju and southern parts of Korea. It is called "Jinju" and it contains phenolic component, triterpene component, and elagic acid component in the outpost. And has been reported to exhibit a spermatic action against human neutrophils in conjunction with P. amarus (Yuen et al, Ilangkovan M, Jantan I, Mohamad HF, Husain K, Abdul Razak AF: Inhibitory effects of standardized extracts of phyllanthus amarus and phyllanthus urinaria and their marker compounds on phagocytic activity of human neutrophils .Evid Based Complement Alternat Med (2013) 2013, pp.603-634 ]. These plants are indigenous to the hot beaches of India and tropical and subtropical countries, and Pilatus amarus has performed clinical trials as a treatment for HIV, jaundice, hypertension and diabetes [Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK: Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology: A review. Journal of ethnopharmacology (2011) 138 (2): pp. 283-3134].

Corilagin; β-1-O- galloyl -3,6- (R) - hexahydroxydiphenoyl -D- glucose) Is a kind of polyphenol which is a kind of hydrolyzable ellagitannins (Zhao L, Zhang SL, Tao JY, Pang R, Jin F, Guo YJ, Dong JH, Ye P, Zhao HY , Zheng GH: Preliminary exploration of anti-inflammatory mechanism of corilagin (beta-1-o-galloyl-3,6- (r) -hexahydroxydiphenoyl-d-glucose) in vitro.International immunopharmacology(2008) 8 (7): 1059-1064; Duan W, Yu Y, Zhang L: Antiatherogenic effects of phyllanthus emblica associated with corilagin and its analogue.Yakugaku zasshi: Journal of the Pharmaceutical Society of Japan (2005) 125 (7): 587-591], plants in 1951Caesalpinia coriaria, ≪ / RTI > Phyllanthus urinaria , Phyllanthus emblica And Phyllanthus amarus (= Phyllanthus niruri )≪ / RTI >Phyllanthus species); (Duan W, Yu Y, Zhang L: Antiatherogenic effects of phyllanthus emblica associated with corilagin and its analogue.Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan (2005) 125 (7): 587-591; Moreira J, Klein-Junior LC, Cechinel Filho V, de Campos Buzzi F: Anti-hyperalgesic activity of corilagin, a tannin isolated from phyllanthus niruri l. (euphorbiaceae).Journal of ethnopharmacology (2013) 146 (1): 318-323; Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK: Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology: A review.Journal of ethnopharmacology (2011) 138 (2): 286-313; Liu KC, Lin MT, Lee SS, Chiou JF, Ren S, Lien EJ: Antiviral tannins from two phyllanthus species.Planta Med (1999) 65 (1): 43-46; Jikai L, Yue H, Henkel T, Weber K: One step purification of corilagin and ellagic acid from phyllanthus urinaria using high-speed counter current chromatography.Phytochem Anal (2002) 13 (1): 1-3). Terminalia catappa L., Nephelium lappaceum L., Alchornea glandulosa , Excoecaria agallocha L.And stamens leafPunica granatum) [Kinoshita S, Inoue Y, Nakama S, Ichiba T, Aniya Y: Antioxidant and hepatoprotective actions of medicinal herb, terminal catapta l. From Okinawa island and its tannin coralagin.Phytomedicine : international journal of phytotherapy and phytopharmacology(2007)14(11): 755-762; Li Y, Yu S, Liu D, Proksch P, Lin W: Inhibitory effects of polyphenols toward hcv from the mangrove plant excoecaria agallocha l. Bioorganic & medicinal chemistry letters (2012)22(2): 1099-1102; Satomi H, Umemura K, Uenoya, Hatano T, Okuda T, Noro T: Carbonic anhydrase inhibitors from the pericarps of punica granatum l. Biological & pharmaceutical bulletin (1993)16(8): 787-790; Thitilertdecha N, Teerawutgulraga, Kilburn JD, Rakariyatham N: Identification of major phenolic compounds from nephelium lappaceum l. And their antioxidant activities.Molecules ( Basel , Switzerland )(2010)15(3): 1453-1465);Terminalia chebula Retz (Park JH, Joo HS, Yoo KY, Shin BN, Kim IH, Lee CH, Choi JH, Byun K Lee B, Lim SS, Kim MJ meat get: Extract from terminalia chebula seeds protect against experimental ischemic neuronal damage via sods and bdnf levels.Neurochemical research (2011)36(11): 2043-2050.] There is a separate bar; Antioxidative effect of corrrelazine, liver protection effect, platelet aggregation inhibitory effect, atherosclerosis activity, antihypertensive effect, anticancer activity (Hau DK, Zhu GY, Leung AK, Wong RS, Cheng GY, Lai PB, Tong SW, Lau FY , Chan KW, Wong WY, Lam KH meat get:In vivo anti - tumour activity of corilagin on Hep3B hepatocellular carcinoma. Phytomedicine: international journal of phytotherapy and phytopharmacology (2010)18(1): 11-15) and anti-hyperalgesic activity are already well known in the prior art.

None of the above documents, however, discloses or discloses the inhibitory activity of fox-marbled extracts and their isolated cholylazine compounds against enteroviruses and coxsackieviruses.

Accordingly, the present inventors have found that the use of Vero cells as a therapeutic agent for enteroviruses and coxsackievirus infections inhibits CVA 16 and EV71 induced cell lesions using fox bead extract and coriolagin isolated therefrom The inventors of the present invention completed the present invention by confirming that the samples of the present invention showed strong antiviral activity against CVA 16 and EV71 virus cell infections and exhibited a strong inhibitory effect on CVA 16 and EV71 induced cell lesions .

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 or coxsackievirus containing fox bead extract or corrillazine isolated therefrom as an active ingredient.

The present invention also provides a health functional food for preventing and ameliorating infectious diseases caused by enterovirus or coxsack virus containing fox marbled extract or corrlazine isolated therefrom as an active ingredient.

The fox bead extract as defined herein is characterized as a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

The crude extract, as defined herein, is a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably water or a mixed solvent of water and methanol, And is an extract which is soluble in methanol.

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 extracted solely in hexane, methylene chloride, chloroform, or ethyl acetate, preferably in 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 or coxsackievirus containing a fox bead extract or a combination of corrillagene isolated therefrom with a conventional anti-enteroviral agent as an active ingredient.

The present invention also provides a health functional food for preventing and ameliorating infectious diseases caused by enterovirus or coxsack virus, which comprises a combination of fox bead extract or corrillagin isolated therefrom with a conventional anti-enteroviral agent as an active ingredient.

Existing anti-enteroviral agents as defined herein are plconaril (Pleconaril, Sigma). Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel) and Rimantadine, Rivaririn, Taribavirin and the like, preferably Pleconaril (Sigma). Oseltamivir or Amantadine.

The enteroviruses as defined herein are serotypes of HEV-A, HEV-B, HEV-C or HEV-D, preferably HEV-A serotypes, more preferably, .

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 fox bead extract of the present invention can be prepared as follows. Dried fox beads 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 methanol, more preferably Is mixed with methanol several times and then subjected to an ultrasonic extraction method, hot water extraction method, room temperature extraction method or reflux extraction method at a temperature of 30 ° C to 150 ° C, preferably 50 ° C to 100 ° C for 30 minutes to 48 hours, preferably 1 hour to 12 hours , 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 the 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 non-polar solvent, such as n-hexane, methylene chloride or ethyl acetate, obtained above, is dissolved in a mixed solvent of hexane and methylene chloride, The purification method using chromatography such as column chromatography, RP C18 column chromatography or silica gel open column chromatography or Diaion HP-20 column chromatography is repeated several times to selectively purify the compound of the present invention, .

The inventors of the present invention found that the samples of the present invention inhibit the CVA 16 and EV71 induced cell lesion inhibition effects and the cytotoxicity test using Vero cells for the Fox bean extract or the compounds isolated therefrom, 16 and EV71 -induced cell lesions and showed strong antiviral activity against CVA 16 and EV71 virus cell infections. Thus, the composition can be used as a pharmaceutical composition for the prevention and treatment of infectious diseases caused by enteroviruses, It was confirmed to be useful as food.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of infectious diseases caused by enterovirus or coxsack virus containing the fox marbled extract obtained by the above production method and the compound isolated therefrom as an active ingredient.

Also, the fox bead leaf has been used for a long time as a herbal medicine, and the compound isolated from the fox bead 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 administration 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, sweetening agents, 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 extract or 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 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 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 enteroviruses containing the fox bean leaf extract 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 fox bead extract of the present invention or the compound isolated therefrom can be used for the test of inhibition of EV71 C3 and EV71 C4a-induced cell lesion using Vero cells and cytotoxicity test Confirmed that the samples of the present invention showed strong inhibitory effects on CVA 16 and EV71 induced cell lesions and showed strong antiviral activity against CVA 16 and EV71 virus cell infections, It is useful as a therapeutic pharmaceutical composition or a health functional food.

FIG. 1 is a graph showing the content analysis of the active compound, corrillazine, by HPLC in ethyl acetate and butanol-soluble fractions, which are active fractions of fox bead leaf extract.
Figure 2 shows anti-viral activity against collagenase EV71 and CVA16;
Figure 3 is a microscopic photograph of the effect on the cytopathic effect of Enterovirus 71 (EV71) induced by (A) non-infected cells, (B) non-infected cells treated with corrrelazine, (C) ribavirin (D) EV71-infected cells, (E) EV71-infected cells treated with collirazin, (F) ribavirin treated EV71-infected cells;
Figure 4 is photographs showing the effect on the cytopathic effect of CVA16 induced cytotoxic effect (A) non-infected cells, (B) non-infected cells treated with corrrelazine, (C) noninflammatory cells treated with ribavirin CVA16-infected cells; (E) CVA16-infected cells treated with corrrelazine; (F) CVA16-infected cells treated with ribavirin;
Figure 5 shows the inhibitory effect of the extracts of the present invention on the CVA16 virus;
6 shows the inhibitory effect of the extracts of the present invention on the EV71 virus.

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   1. Preparation of fox bead extract

1-1. Fox beads Crude extract  Produce

Fox beads ( Phyllanthus dried leaves and branches 600g (Yonsei sample number of urinaria); YEPU-1310) 3 times ultrasonic extractor (8510 DHT for 3 hours at room temperature, was added to the methanol solvent 2L on; extracted with CT, USA), and then vacuum filtered and the supernatant Respectively. This process 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 95.4 g of methanol extracts of fox beads (hereinafter referred to as "PUM" ).

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

The dried methanol extract obtained in Example 1-1 was suspended in 500 ml of distilled water and subjected to fractionation using n-hexane, chloroform, ethylacetate and n-butanol (BuOH) according to a conventional fractionation method , And dried under reduced pressure to obtain 3.9 g of hexane-soluble fraction, 6.4 g of chloroform, 21.4 g of ethyl acetate-soluble fraction, 39.2 g of butanol-soluble fraction and 24.3 g of water-soluble fraction, and lyophilized (hereinafter referred to as "PUHE" PUCH "," PUEA "," PUBU ", and" HH-WA ").

Example   2. Fox ball  Isolation of the active compound from the extract

For the identification of the active substance, 21.4 g, 39.2 g of the ethyl acetate-soluble fraction and the butanol-soluble fraction in the most active fractions in the following experiment were subjected to HPLC-DAD (Agilent 1290 HPLC) system and YMC-Hydrosphere C ₁₈ × 150 mm ID) column chromatography was carried out under the following analytical conditions, and the active ingredient, corrillazine, in the ethyl acetate-soluble fraction and butanol-soluble fraction was quantitatively analyzed by comparison with a standard product (Corilaginl, provided by Professor Kim Seunghyun of Yonsei University).

HPLC analysis conditions Reference UV Detector 254 nm Mobile phase (A) acetonitrile and (B) water with 0.1% phosphoric acid Flow rate 1 mL / min

As a result of the above analysis, a standard calibration curve for cholylazine was obtained. Coriolus concentrations of the EtOAc and n- BuOH soluble fractions of fox beads were 8.7% and 5.6%, respectively, This means that corrrelazine is a major component of the fractions (Figure 1).

Reference Example 1. Viruses, cell lines and reagents

The CVA16 and EV71 viruses were obtained from the Korea Center for Disease Control and Prevention (KCDC), and vero cells isolated from African green monkey kidney (CCL-81, ATCC) Lt; / RTI > Vero cells were maintained in MEM media (11095-080, Gibco) supplemented with 10% FBS (16000-044, Gibco) and 0.01% antibiotic (15070-063, Gibco). Antibiotic-antimycotic solution, trypsin-EDTA, FBS and MEM media were purchased from Gibco BRL (Invitrogen Life Technologies, Karlsruhe, Germany). Tissue culture plates were purchased from Falcon (BD Biosciences, San Jose, CA, USA) and sulforhodamine B (SRB) purchased from SigmaAldrich (S9012-25G). All other reagents were reagent grade.

Reference example  2. Statistics

All test results were expressed as mean ± SD and the statistical significance used was one-way ANOVA with Tukey's post hoc test. Values of p <0.05, p <0.01, and p <0.001 were considered statistically significant.

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 a number 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, 48 hours after infection, 0.09 ml of diluted virus suspension containing 50% cell culture infectious dose (CCID ₅₀ ) was added to produce appropriate CPE and 0.01 ml of FBS supplemented medium containing various concentrations of sample was added Respectively. The antiviral activity of each sample was measured at a 5-fold step-wise dilution ranging from 0.4 to 50 μg / mL. Four wells were used as virus-infected non-compound-treated cells without compound treatment. Four other wells were used as non-infected non-infected cells -compound-treated cells). The culture plates were cultured in a CO ₂ incubator (VS-9108MS, Vision, Korea) at 37 ° C and 5% CO ₂ for 2 days until appropriate CPE was achieved. Washed once, and 100 μL 70% ice-cold acetone was added to each well and left at 20 ° C for 30 minutes. 70% acetone was removed, the plate was dried in a drying oven for 30 minutes, and 100 μL of SRB staining reagent in which 0.4% (w / v) SRB was dissolved in 1% acetic acid solution was added to each well and left at room temperature for 30 minutes. The SRB was removed and the plate was washed five times with 1% acetic acid before oven-drying. After drying the 96-well plate for 1 day in a drying oven, the morphology of the cells was measured by a phase contrast microscope (Axiovert 10; Zeiss, Wetzlar, Germany) in order to observe the effect of the samples on CVA 16 and EV71 induced CPE. Images were recorded after observing at 10 magnification. Each well dyed with the SRB staining reagent was dissolved in 100 mM of a 10 mM unbuffered Tris-base solution and the plate was left on the table for 30 minutes. The absorbance was read at absorbance (562 nm) using a reader (VERSAmax microplate reader; Molecular Devices, Palo Alto, Calif., USA) with a comparative absorbance value of 620 nm. The results were then converted to the percent protection by the test CFS in EV71-infected cells and by the following equation (1).

(Expressed in%)

Where (OD _t ) _EV71 is the optical density due to the given CFS in _EV71- infected cells;

(OD _c ) _EV71 is the optical density by control untreated EV71-infected cells in control untreated EV71- infected cells;

And (OD _c ) _mock Lt; / RTI &gt; is the optical density by control mock-infected cells.

The antiviral activity was expressed as% (% of control) of the control. Ribavirin (Duchefa, R1082) and DMSO were used as positive and negative controls, respectively.

1-2. Cytotoxicity test

The cells were inoculated into 96-well plates at a concentration of 2 × 10 ⁴ cells / well. The medium was removed the next day and washed with PBS. Each Vero cell was treated with a virus-infected cell culture in a maintenance medium and incubated in a CO ₂ incubator at 37 ° C for 2 days. For each sample, 3 wells were used as control without sample treatment, cytotoxicity was evaluated by SRB assay, and cytotoxicity was expressed as% of control (Choi HJ , Song JH, Park KS, Kwon DH: Inhibitory effects of quercetin 3-rhamnoside on influenza virus replication. Eur J Pharm Sci (2009) 37 (3-4): 329-333)

The antiviral activity and cytotoxicity of the organic solvent-soluble fractions, including methanol extracts and EtOAc and BuOH soluble fractions, were measured at a 5-fold step-wise dilution in the 0.4 to 50 μg / mL range.

Experiment result

1-3. Correlazin EV71  And CVA16  Antiviral activity against viruses

As a result of this experiment, Corradil showed strong antiviral activity against EV71 and CVA16. In the case of CVA16 infection, the 50 μg / ml ribavirin treatment did not increase the cell survival rate compared with the DMSO treatment control group. However, CVA16 infection did not increase the survival rate of CVA16 infection. However, CVA16 infection by 50 μg / ml and 10 μg / 65% and 38% of Vero cells survived. (Fig. 2A). Similarly, in the case of EV71 infection, only 50 μg / ml ribavirin treatment significantly increased the cell survival rate, but the cell viability was estimated to be about 63% of the EV71 infected cells by 10 μg / ml Coriolus treatment (Fig. 2B). In both the CVA16 and EV71 infection studies, ribavirin exhibited a marginal antiviral activity of less than 50% at a concentration of 50 μg / ml, whereas the IC ₅₀ value, which indicates a 50% inhibitory effect on the viiral activity of cholylazine, And 5.60 ± 1.07 μg / ml and 32.33 ± 0.42 μg / ml for the EV71 and CVA16 viruses, respectively. (Table 1). In conclusion, corrrelazine has shown strong antiviral activity against CVA 16 and EV71 infections in Vero cells.

Antiviral activity of corrrelazine against CVA16 and EV71 CVA16 EV71 Compound CC ₅₀ ^a IC ₅₀ ^b TI ^c CC ₅₀ ^a IC ₅₀ ^b TI ^c Corilagin 107 32.330.42 3.30 327 5.601.07 58.39 Ribavirin > 50 ND ^d - > 50 ND ^d - Results are presented as the mean IC ₅₀ values ± SD obtained from three independent experiments carried out in triplicates.
^a Concentration required to reduce cell growth by 50% (μg / mL).
^b Concentration required to inhibit virus-induced CPE by 50% (μg / mL).
^c Therapeutic index = CC ₅₀ / IC ₅₀
^d Not determined

1-4. Correlazin CVA16 - and EV71 - induced cell degeneration ( induced cytopathic )

In order to confirm the cytoprotective effect of corrillazine on CVA16 and EV71 infection, the morphology of Vero cells treated with the sample after CVA16 or EV71 infection was confirmed.

In the absence of infection by CVA16 and EV71 in vero cells, vehicle-treated vero cells exhibited a typical spread-out and normal morphology (Figures 3A and 4A ). In addition, cells treated with 50 μg / mL corilagin (FIGS. 3B and 4B) or ribavirin (FIGS. 3C and 4C) were not altered morphologically and cytotoxicity was not found. BERO cells infected with CVA16 or EV71 without corrillagen resulted in severe cellular lesions (FIGS. 3D and 4D), whereas collillazin treatment of infected BERO cells inhibited the cell lesion shown (Figures 3E and 4E). On the other hand, the treatment with ribavirin after infection with CVA16 and EV71 in Vero cells showed only a slight inhibition of CPE (Figs. 3F and 4F). Thus, the cell lesions of CVA16 or EV71 virus-infected Bero cells were inhibited in the presence of corrrelazine.

Also, the methanol extract (PUM) is inhibited by CVA16 (Figure 5) and, respectively 45.9 ± 5.39 μg / mL and 32.4 ± 0.35 μg / mL in EV71 number (Fig. 6) the IC ₅₀ 80% or more teeth (Table 2). Ethyl acetate and butanol-soluble fractions also showed antiviral activity at a concentration of 50 μg / ml of 4.79 ± 0.81 μg / mL and 39.5 ± 6.51 μg / mL, respectively, on the IC ₅₀ surface of the CVA16 of about 50% or more And Table 2). Further, ethyl acetate and butanol soluble fraction In Figure inhibition, IC ₅₀ can tooth surface of about 51% and 95% with respect to the EV71 at 50 μg / ml concentrations respectively to 43.8 ± 9.17 μg / mL, and 31.8 ± 2.29 μg / mL Indicating antiviral activity (Table 2). It was confirmed that the ethyl acetate and butanol soluble fractions of corrlazin and fox beads containing the cholylazine and its main component exhibited strong antiviral activity against CVA16 and EV71, and it was confirmed that antiviral therapeutic agents useful for the development of HFMD therapy could be produced .

Antiviral activity of fox bead extracts on CVA16 and EV71 in Vero cells CVA16 EV71 Test material CC ₅₀ ^a IC ₅₀ ^b TI ^c CC ₅₀ ^a IC ₅₀ ^b TI ^c Methanol > 50 45.9 ± 5.39 2.17 > 50 32.4 ± 0.35 1.54 Hexane > 50 ND ^d - > 50 ND ^d - Ethyl acetate > 50 4.79 ± 0.81 17.2 > 50 48.3 ± 9.17 1.41 Butanol > 50 39.5 ± 6.51 2.53 > 50 31.8 ± 2.29 1.57 Water > 50 ND ^d - > 50 ND ^d - Ribavirin > 50 ND ^d - > 50 ND ^d - Results are presented as the mean IC ₅₀ values ± SD obtained from three independent experiments carried out in triplicates.
^a Concentration required to reduce cell growth by 50% (μg / mL).
^b Concentration required to inhibit virus-induced CPE by 50% (μg / mL).
^c Therapeutic index = CC ₅₀ / IC ₅₀
^d Not determined

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, methanol extract of fox beads, ethyl acetate fraction, butanol fraction and cholylazine did not show acute toxicity until oral administration of 2 g / kg.

As a result of the above experiment, fox beads extract, fractions and collillazin can safely be used for infuenza virus 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

<Formulation Example 1-1>

PUM ------------------------------------------------- - 300 mg

D-mannitol --------------------------------------------- 100 mg

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

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

<Formulation Example 1-2>

PUM ------------------------------------------------- - 300 mg

D-mannitol --------------------------------------------- 100 mg

Glyceryl behenate ------------------------------------- 10 mg

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

Formulation Example 2. Preparation of tablets

<2-1. Direct Tacking Method>

PUEA ------------------------------------------------- - 300 mg

Microcrystalline cellulose --------------------------------------- 75 mg

Di-mannitol ---------------------------------------------- 75 mg

Croscarmellose sodium --------------------------------- 18 mg

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

After mixing the above components, the tablets are prepared by tableting according to a conventional tablet preparation method, followed by coating with the following excipients to prepare tablets.

Polyvinyl alcohol ----------------------------------------- 6.83 mg

Titanium oxide --------------------------------------------- 4.80 mg

Talk ------------------------------------------------- 3.00 mg

Lecithin ----------------------------------------------- 0.30 mg

Xanthan gum ----------------------------------------------- 0.07 mg

<2-2 Wet Granulation Method>

PUEA ------------------------------------------------- - 300 mg

Microcrystalline cellulose --------------------------------------- 75 mg

Lactose hydrate --------------------------------------------- 75 mg

Hydroxypropylcellulose 5 mg &lt; RTI ID = 0.0 &gt;

Croscarmellose sodium --------------------------------- 18 mg

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

After the preparation of the binding liquid, the above components are mixed and the tablets are prepared through a wet granulation method according to a conventional tablet preparation method, and the tablets are prepared by coating with the following excipients after coating liquid preparation.

Polyvinyl alcohol ----------------------------------------- 6.83 mg

Titanium oxide --------------------------------------------- 4.80 mg

Talk ------------------------------------------------- 3.00 mg

Lecithin ----------------------------------------------- 0.30 mg

Xanthan gum ----------------------------------------------- 0.07 mg

Formulation example  3. Preparation of capsules

<3-1.>

PUBU ------------------------------------------------- 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.

<3-2. Prescription>

PUBU ------------------------------------------------- 300 mg

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

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

Croscarmellose sodium --------------------------------- 5 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 injection

Correlazin compound - 300 mg

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

Sterile sterile distilled water used - 297 mg

Na ₂ HPO ₄ 12H ₂ O ------------------------------------------- - 26 mg

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

Formulation Example 5. Preparation of a liquid preparation

In this about 100 mL

PUHE ------------------------------------------------- 1500 mg

Citric acid ---------------------------------------------- 200 mg

Sodium citrate hydrate ---------------------------------- 200 mg

DL-malic acid --------------------------------------------- 300 mg

Sucralose ------------------------------------------ 100 mg

White Party ------------------------------------------------- --- 15 g

D-sorbitol solution -------------------------------------------- 15 g

Polysorbate 80 --------------------------------------- 30 mg

Xanthan gum ------------------------------------------------ - 70 mg

P-hydroxybenzoic acid methyl 30 mg &lt; RTI ID = 0.0 &gt;

P-hydroxybenzoic acid propyl 30 mg

Flavoring agent ------------------------------------------------ - Suitable amount

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

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved. After mixing the above components, purified water was added, a proper amount of flavoring agent was added, and purified water was added to adjust the total volume to 100 ml. To prepare a liquid agent.

Formulation Example 6. Preparation of Healthy Foods

Corrylazine Compound 1000 mg &lt; RTI ID = 0.0 &gt;

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

Vitamin A Acetate ---------------------------------- 70 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 μg

Nicotinic acid amide 1.7 mg

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

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

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

Ferrous sulfate ------------------------------------------ 1.75 mg

Zinc oxide - 0.82 mg

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

Potassium phosphate monohydrate 15 mg

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

PUCH ------------------------------------------------ 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 ------------------------------------------------- --- Quantity

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 (13)

A pharmaceutical composition for the prophylaxis and treatment of infectious diseases caused by enterovirus or coxsackievirus containing fox bead extract or corrrelazine isolated therefrom as an active ingredient. The method according to claim 1,
Wherein the fox bead 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. A pharmaceutical composition for the prophylaxis and treatment of infectious diseases caused by enterovirus or coxsackievirus containing a fox bead extract or a combination of corrillagen isolated therefrom and an existing anti-enteroviral agent as an active ingredient. The method according to claim 6,
The conventional anti-enteroviral agent is Pleconaril (Sigma). Wherein the composition is selected from Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel) and Rimantadine, Rivaririn, or Taribavirin. 7. The method according to claim 1 or 6,
Wherein said enterovirus is a serotype of HEV-A, HEV-B, HEV-C, or HEV-D. 7. The method according to claim 1 or 6,
Wherein said enterovirus is characterized by a serotype of coxsakiovirus A16 type, enterovirus type 71. 7. The method according to claim 1 or 6, wherein the infectious disease caused by the enterovirus or the coxsack virus is selected from the group consisting of conjunctivitis, otitis media, skin rash, aseptic meningitis, herpetic sinusitis, Encephalitis, Guillian-Barre syndrome, ataxia, terminal neuritis, transverse myelitis, limb paralysis, diabetes mellitus or epidemic hemorrhagic keratoconjunctivitis. A health functional food for preventing and ameliorating infectious diseases caused by enterovirus or coxsack virus containing an extract of fox beads or corrrelazine isolated therefrom as an active ingredient. A health functional food for preventing and ameliorating infectious diseases caused by enterovirus or coxsackievirus containing a fox bead extract or a combination of corrillagen isolated therefrom and an existing anti-enteroviral agent as an active ingredient. 13. The method according to claim 11 or 12,
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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