Manuscript to be reviewed Ficus septica plant extracts for treating Dengue virus in vitro Nan-Chieh Huang 1 , Wan-Ting Hung 2 , Wei-Lun Tsai 3 , Feng-Yi Lai 4 , You-Sheng Lin 5 , Mei-Shu Huang 5 , Jih-Jung Chen 6 , Wei-Yu Lin 7 , Jing-Ru Weng Corresp., 8 , Tsung-Hsien Chang Corresp. 5, 9 1 Department of Family Medicine, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan 2 Section of critical care medicine, Kaohsiung Veteran General Hospital, Kaohsiung, Taiwan 3 Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 4 Deparment of Nursing, Shu-Zen College of Medicine and Management, Kaohsiung, Taiwan 5 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 6 Department of Pharmacy, Tajen University, Pingtung, Taiwan 7 Department of Pharmacy, Kinmen Hospital, Kinmen, Taiwan 8 Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung, Taiwan 9 Department of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan Corresponding Authors: Jing-Ru Weng, Tsung-Hsien Chang Email address: [email protected], [email protected] Dengue virus type 1- 4 (DENV-1-4) are positive-strand RNA viruses with an envelope that belong to the Flaviviridae. DENV infection threatens human health worldwide. However, other than supportive treatments, no specific therapy is available for the infection. To discover novel medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plants in vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves and stem from Ficus septica Burm. f. had a promising anti-DENV-1 and DENV-2 effect. However, infection with the non-envelope picornavirus, Aichi virus, was not inhibited by treatment with F. septica extracts. F. septica may be a candidate antiviral drug against an enveloped virus such as DENV. PeerJ reviewing PDF | (2016:11:14751:2:0:NEW 19 May 2017) Manuscript to be reviewed 1 Ficus septica plant extracts for treating Dengue virus in vitro 2 3 Nan-Chieh Huang1,#, Wan-Ting Hung2,#, Wei-Lun Tsai3,#, Feng-Yi Lai4, You-Sheng Lin5, Mei- 4 Shu Huang5, Jih-Jung Chen6, Wei-Yu Lin7, Jing-Ru Weng8,* and Tsung-Hsien Chang5,9* 5 6 1 Department of Family Medicine, Zuoying Branch of Kaohsiung Armed Forces General 7 Hospital, Kaohsiung, Taiwan 8 2 Section of critical care medicine, Kaohsiung Veteran General Hospital, Kaohsiung, Taiwan 9 3 Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Veterans General 10 Hospital, Kaohsiung, Taiwan 11 4 Deparment of Nursing, Shu-Zen College of Medicine and Management, Kaoshiung, Taiwan 12 5 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, 13 Kaohsiung, Taiwan 14 6 Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung, Taiwan 15 7 Department of Pharmacy, Kinmen Hospital, Kinmen, Taiwan 16 8 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, 17 Kaohsiung, Taiwan 18 9Department of Medical Laboratory Science and Biotechnology, Chung Hwa University of 19 Medical Technology, Tainan, Taiwan 20 21 # These authors contributed equally to this work. 22 *Corresponding authors, E-mail: [email protected] (T.-H. Chang) or 23 [email protected] (J.-R. Weng) 24 PeerJ reviewing PDF | (2016:11:14751:2:0:NEW 19 May 2017) Manuscript to be reviewed 25 Abstract 26 Dengue virus type 1- 4 (DENV-1-4) are positive-strand RNA viruses with an envelope that 27 belong to the Flaviviridae. DENV infection threatens human health worldwide. However, other 28 than supportive treatments, no specific therapy is available for the infection. To discover novel 29 medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plants in 30 vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves 31 and stem from Ficus septica Burm. f. had a promising anti-DENV-1 and DENV-2 effect. 32 However, infection with the non-envelope picornavirus, Aichi virus, was not inhibited by 33 treatment with F. septica extracts. F. septica may be a candidate antiviral drug against an 34 enveloped virus such as DENV. 35 PeerJ reviewing PDF | (2016:11:14751:2:0:NEW 19 May 2017) Manuscript to be reviewed 36 Introduction 37 Dengue fever is an acute infectious disease caused by dengue virus (DENV), which is 38 transmitted by mosquitoes to humans, about 50 million people are infected per year worldwide 39 (Guzman et al., 2010). According to different serotypes of the virus, four types (DENV type 1-4) 40 are divided. Each type has the ability to cause disease. DENV infection causes varying degrees 41 of disease manifestation, such as self-limited febrile dengue fever, skin rash or drowsiness, 42 agitation, liver enlargement, or dengue hemorrhagic fever (DHF) and even death. A second 43 DENV infection may lead a life-threatening dengue shock syndrome (DSS) (Abel et al., 2012; 44 Kyle and Harris, 2008; Martina et al., 2009). Currently, no specific therapy is available for the 45 infection other than supportive treatments (Guzman et al., 2010). 46 The identification and use of medicinal plants for treatment of various diseases is 47 throughout human history. Certain medicinal plants also show antiviral activity, such as Carissa 48 edulis Vahl against herpes simplex virus, Geranium sanguineum L. against influenza virus A, 49 Boehmeria nivea L. against hepatitis B virus, Saxifraga melanocentra Engl. & Irmsch. against 50 hepatitis C virus, Lycoris radiata (L'Hér.) Herb. against severe acute respiratory syndrome- 51 associated coronavirus and Phyllanthus amarus Schum. & Thonn. against HIV (Mukhtar et al., 52 2008). In addition, the neem (Azadirachta indica A. Juss.) showed potential inhibition of DENV- 53 2 replication (Parida et al., 2002). Thus, discovering a novel antiviral medicine from medical PeerJ reviewing PDF | (2016:11:14751:2:0:NEW 19 May 2017) Manuscript to be reviewed 54 plants would be a promising strategy. 55 In this study, we collected 23 plants among Taiwanese folk medicinal plants for screening 56 anti-DENV herbs. We also investigated the antiviral effect on Aichi virus (AiV), a pathogenic 57 picornavirus that causes gastroenteritis. Among these plants, we found that F. septica Burm. f. 58 could be a potential medicinal plant against DENV. 59 60 Materials and methods 61 Virus and cell line 62 We used local Taiwanese strains of DENV-1 766733A and DENV-2 PL046 (Genbank accession 63 no. AJ968413.1) isolated from patients with dengue fever (Lin et al., 1998). The viruses were 64 propagated in mosquito cell line C6/36 (ATCC: CRL-1660) grown in RPMI 1640 medium 65 containing 5% fetal bovine serum (FBS). The human Aichi virus (AiV) was isolated from a 66 newborn with diarrhea in Taiwan and propagated in Vero cells (ATCC: CCL-81) (Chen et al., 67 2013). Vero cell, A549 human lung epithelial carcinoma cells (ATCC: CCL-185) and Huh7.5 68 human hematoma cells (ATCC® PTA-8561™) were cultured in DMEM supplemented with 10% 69 fetal bovine serum (FBS; Thermo Fisher). HepG2 human hepatocellular carcinoma cells 70 (ATCC® HB-8065™) and WS1 human fetal skin normal fibroblasts (BCRC: 60300) were PeerJ reviewing PDF | (2016:11:14751:2:0:NEW 19 May 2017) Manuscript to be reviewed 71 cultured in MEM medium supplemented with 10% FBS and non-essential amino acids (NEAA, 72 Gibco, Thermo Fisher). 73 74 Plant materials 75 All plants were purchased from traditional herb shop or Chinese medicinal herb shop in Taiwan. 76 The plants were identified by one of the co-authors, Dr. Wei-Yu Lin (October, 2008 to May, 77 2014). Those plants are Alisma orientalis (Sam.) Juz., Asparagus cochinchinensis (Lour.) Merr., 78 Broussonetia papyrifera (L.) LHerit. ex Vent., Catharanthus roseus (L.) G. Don, Clausena 79 excavate Burm. f., Cinnamomum insulari-montanum Hayata, Cornus officinalis Torr. ex Dur., 80 Euonymus japonicas Thunb., Elaeocarpus sylvestris (Lour.) Poir., Fraxinus griffithii C. B. 81 Clarke, Ficus septica, Ficus sarmentosa B. Ham. ex J. E. Sm. var. henryi (King ex D. Oliver) 82 Corner, Garcinia subelliptica Merr., Lumnitzera racemosa Willd., Litchi chinensis Sonn., 83 Phytolacca americana L., Pueraria lobata (Willd.) Ohwi ssp. thomsonii (Benth.) Ohashi & 84 Tateishi, Sida acuta Burm. f., Sambucus chinensis Lindl., Scrophularia ningpoensis Hemsl, 85 Saurauia tristyla var. oldhamii (Hemsl.) Finet & Gagnep., Tribulus terrestris L., Xanthium 86 sibiricum Patrin ex Widder and Strophanthus divaricatus (Lour.) Hook. Edt Arn. We included 87 material from the whole plant, root, leaves, stem, fruit, pericarp, root bark, flower or heartwood. 88 PeerJ reviewing PDF | (2016:11:14751:2:0:NEW 19 May 2017) Manuscript to be reviewed 89 Plant extracts 90 Materials of plant species were ground, extracted with the indicated solvent for 1 week. The 91 extracts were concentrated under vacuum. Plant materials of the species no. 13 (leaves) was 92 subjected to an additional one-step extraction with ethyl acetate (EtOAc) and filtered and dried 93 as described before. The volumes (5 ml) of solvents were used per gram of plant material. 94 95 Cell proliferation assay 96 WST-1 assay (Roche, Basel, Switzerland) was used to monitor cell proliferation (Chou et al., 97 2014); cells were trypsinized and resuspended in culture medium, then plated at 5×103 cells per 98 well in 96-well plates and incubated overnight. After plant extracts treatment for 48 h, the cells 99 were incubated with 10 μl WST-1 reagent for 2 h. The cell viability was quantified by multi-well 100 spectrophotometry (Anthos, Biochrom, Cambridge, UK). The absorbance at 450 nm was 101 monitored, and the reference wavelength was set at 620 nm. 102 103 Treatment 104 In the extracts screening of DENV inhibition, the cells (5×103 cells) were treated with plant 105 extracts with serial dilution dose of 100, 50, 25, 12.5, 6.25, 3.125 or 1.56 g/ml or DMSO 106 solvent control for 3 h.