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      Eupafolin and Ethyl Acetate Fraction of Kalanchoe gracilis Stem Extract Show Potent Antiviral Activities against Enterovirus 71 and Coxsackievirus A16

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          Abstract

          Enterovirus 71 (EV71) and coxsackievirus A16 (CoxA16) are main pathogens of hand-foot-and-mouth disease, occasionally causing aseptic meningitis and encephalitis in tropical and subtropical regions. Kalanchoe gracilis, Da-Huan-Hun, is a Chinese folk medicine for treating pain and inflammation, exhibiting antioxidant and anti-inflammatory activities. Our prior report (2012) cited K. gracilis leaf extract as moderately active against EV71 and CoxA16. This study further rates antienteroviral potential of K. gracilis stem (KGS) extract to identify potent antiviral fractions and components. The extract moderately inhibits viral cytopathicity and virus yield, as well as in vitro replication of EV71 (IC 50 = 75.18  μ g/mL) and CoxA16 (IC 50 = 81.41  μ g/mL). Ethyl acetate (EA) fraction of KGS extract showed greater antiviral activity than that of n-butanol or aqueous fraction: IC 50 values of 4.21  μ g/mL against EV71 and 9.08  μ g/mL against CoxA16. HPLC analysis, UV-Vis absorption spectroscopy, and plaque reduction assay indicate that eupafolin is a vital component of EA fraction showing potent activity against EV71 (IC 50 = 1.39  μ M) and CoxA16 (IC 50 = 5.24  μ M). Eupafolin specifically lessened virus-induced upregulation of IL-6 and RANTES by inhibiting virus-induced ERK1/2, AP-1, and STAT3 signals. Anti-enteroviral potency of KGS EA fraction and eupafolin shows the clinical potential against EV71 and CoxA16 infection.

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          Outbreaks of hand, foot, and mouth disease by enterovirus 71. High incidence of complication disorders of central nervous system.

          S Nakano, S. Takami, K. Yamaoka … (1980)
          In Japan we have had two outbreaks of hand, foot, and mouth disease associated with disorders of the central nervous system, one in 1973 and the other in 1978. The isolated virus in both outbreaks was enterovirus 71. Central nervous system disorders were present in 24% of patients in 1973 and in 8% of patients in 1978. These disorders were localised encephalitis with cerebellar signs as the main feature, aseptic meningitis, and polio-like paresis. The enterovirus 71 isolated in Japan had strong dermatotropic as well as neurotropic tendencies. However in cross-neutralisation tests, no difference in antigenicity from the prototype, BrCr strain, was recognised.
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            Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India.

            V Gopalkrishna, Shobha Chitambar, Vijay Patil (2012)
            Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, can be caused by enteroviruses. Coxsackievirus A16 (CV-A16) and enterovirus 71(EV-71) are the major aetiological agents of HFMD. Other EV serotypes, CV-A4-7, CV-A9-10, CV-B1-3, CV-B5, E-4 and E-19, have also been found associated with both sporadic infections and outbreaks of HFMD. In India, outbreaks of HFMD have been documented; however, molecular characterization of the aetiological agents has rarely been reported. Cases of HFMD were identified during 2009-2010 on the basis of clinical features in southern and eastern parts of India. The aim of the present study was to detect and characterize the aetiological agents associated with the disease. A total of 89 specimens consisting of 41 sera, 24 vesicular fluids, 18 stools and 6 throat swabs were collected from 61 clinically diagnosed HFMD cases from southern and eastern parts of India. RT-PCR followed by sequencing of PCR amplicons and phylogenetic analysis were performed on all specimens for detection of EV RNA and identification of EV types. EV RNA was detected in 47.1 % (42/89) of the specimens collected from 57.4 % (35/61) of the HFMD cases. Thirty-six of 42 EV strains showed amplification of the VP1/2A junction or VP1 regions. Sequence analysis of the amplicons identified the presence of CV-A16 (54.8 %), CV-A6 (38.1 %), EV-71 (2.4 %), CV-A10 (2.4 %) and E-9 (2.4 %) serotypes in the HFMD cases. The study documents CV-A16 and CV-A6 as major and CV-A10, EV-71 and E-9 as rare viral pathogens of HFMD in India.
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              Aloe-emodin is an interferon-inducing agent with antiviral activity against Japanese encephalitis virus and enterovirus 71

              Cheng-Wen Lin, Chia-Fang Wu, Nai-Wan Hsiao … (2008)
              In this study, aloe-emodin was identified as a potential interferon (IFN)-inducer by screening compounds from Chinese herbal medicine. Aloe-emodin showed low cytotoxicity to human HL-CZ promonocyte cells and TE-671 medulloblastoma cells and significantly activated interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS)-driven cis-reporting systems. Moreover, aloe-emodin upregulated expression of IFN-stimulated genes such as dsRNA-activated protein kinase and 2′,5′-oligoisoadenylate synthase. Aloe-emodin resulted in significant activation of nitric oxide production. The antiviral activity of aloe-emodin against Japanese encephalitis virus (JEV) and enterovirus 71 (EV71) was evaluated using dose- and time-dependent plaque reduction assays in HL-CZ cells and TE-671 cells. The 50% inhibitory concentration (IC50) of aloe-emodin ranged from 0.50 μg/mL to 1.51 μg/mL for JEV and from 0.14 μg/mL to 0.52 μg/mL for EV71. Aloe-emodin showed clearly potent virus inhibitory abilities and achieved high therapeutic indices, in particular for HL-CZ cells. Therefore, the study demonstrated dose- and time-dependent actions of aloe-emodin on the inhibition of JEV and EV71 replication via IFN signalling responses.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Evid Based Complement Alternat Med
                Journal ID (iso-abbrev): Evid Based Complement Alternat Med
                Journal ID (publisher-id): ECAM
                Title: Evidence-based Complementary and Alternative Medicine : eCAM
                Publisher: Hindawi Publishing Corporation
                ISSN (Print): 1741-427X
                ISSN (Electronic): 1741-4288
                Publication date (Print): 2013
                Publication date (Electronic): 2 September 2013
                Publication date PMC-release: 2 September 2013
                Volume: 2013
                Electronic Location Identifier: 591354
                Affiliations
                1School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan
                2Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan
                3School of Pharmacy, China Medical University, Taichung 404, Taiwan
                4Department of Nursing, Hungkuang University, Taichung 433, Taiwan
                5Institute of Biochemistry, National Chung Hsing University, Taichung 402, Taiwan
                6Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming University, Taipei 112, Taiwan
                7Fujian Center for Disease Control and Prevention, Fuzhou, Fujian 350001, China
                8Department of Biotechnology, Asia University, Wufeng, Taichung 413, Taiwan
                Author notes
                *Yuan-Shiun Chang: yschang@ 123456mail.cmu.edu.tw and

                Academic Editor: Shun-Wan Chan

                Article
                DOI: 10.1155/2013/591354
                PMC ID: 3775429
                PubMed ID: 24078828
                SO-VID: 21520c65-c59e-486d-896b-9cb815875b87
                Copyright © Copyright © 2013 Ching-Ying Wang et al.
                License:

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 17 April 2013
                Date accepted : 24 July 2013
                Categories
                Subject: Research Article

                ScienceOpen disciplines: Complementary & Alternative medicine
                Data availability:
                ScienceOpen disciplines: Complementary & Alternative medicine

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