Antiviral Activity of Sambucus Formosananakai Ethanol Extract and Related Phenolic Acid Constituents Against Human Coronavirus NL63 T
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Virus Research 273 (2019) 197767 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Antiviral activity of Sambucus FormosanaNakai ethanol extract and related phenolic acid constituents against human coronavirus NL63 T Jing-Ru Wenga, Chen-Sheng Linb,1, Hsueh-Chou Laic,d,1, Yu-Ping Line, Ching-Ying Wange, ⁎ Yu-Chi Tsaie, Kun-Chang Wuf, Su-Hua Huangg, Cheng-Wen Line,g,h, a Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan b Division of Gastroenterology, Kuang Tien General Hospital, Taichung, Taiwan c School of Chinese Medicine, China Medical University, Taichung, Taiwan d Division of Hepato-gastroenterology, department of internal medicine, China Medical University Hospital, Taichung, Taiwan e Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan f School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan g Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan h Chinese Medicine Research center, China Medical University, Taichung, Taiwan ARTICLE INFO ABSTRACT Keywords: Human coronavirus NL63 (HCoV-NL63), one of the main circulating HCoVs worldwide, causes respiratory tract Human coronavirus NL63 illnesses like runny nose, cough, bronchiolitis and pneumonia. Recently, a severe respiratory illness outbreak of Sambucus FormosanaNakai HCoV-NL63 has been reported in a long-term care facility. Sambucus FormosanaNakai, a species of elderberry, is a ff Ca eic acid traditional medicinal herb with anti-inflammatory and antiviral potential. The study investigated the antiviral Antiviral activity of Sambucus FormosanaNakai stem ethanol extract and some phenolic acid constituents against HCoV- Virus yield NL63. The extract was less cytotoxic and concentration-dependently increased anti-HCoV-NL63 activities, in- Attachment inhibition cluding cytopathicity, sub-G1 fraction, virus yield (IC50 = 1.17 μg/ml), plaque formation (IC50 = 4.67 μg/ml) and virus attachment (IC50 = 15.75 μg/ml). Among the phenolic acid constituents in Sambucus FormosanaNakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity that was ranked in the following order of virus yield reduction: caffeic acid (IC50 = 3.54 μM) > chlorogenic acid (IC50 = 43.45 μM) > coumaric acid (IC50 = 71.48 μM). Caffeic acid significantly inhibited the replication of HCoV-NL63 in a cell-type independent manner, and specifically blocked virus attachment (IC50 = 8.1 μM). Therefore, the results revealed that Sambucus Formosana Nakai stem ethanol extract displayed the strong anti- HCoV-NL63 potential; caffeic acid could be the vital component with anti-HCoV-NL63 activity. The finding could be helpful for developing antivirals against HCoV-NL63. 1. Introduction into nonstructural proteins (nsps) that modulate in viral RNA replica- tion. Among common human coronaviruses like HCoV-229E, HCoV- Human coronavirus (HCoV) NL63, the member of the genus alpha- HKU1, and HCoV-OC43, HCoV-NL63 is one of main circulating HCoVs coronavirus in the Coronaviridae family, is one of common human cor- in the fall and winter worldwide, causing mild upper respiratory tract onaviruses (Li and Lin, 2013; Huang et al., 2017). HCoV-NL63 genome illnesses like runny nose, cough and sore throat in young children, is a positive-strand RNA with near 27.5 kb nucleotides containing 5′ young adults and elderly (Cui et al., 2011; Dijkman et al., 2012). In- untranslated regions (UTR), ORF1a/b, spike(S), ORF3, envelope(E), terestingly, HCoV-NL63 has been frequently detected than other membrane(M), nucleoprotein (N), and 3′UTR (Geng et al., 2012). HCoVs, influenza viruses, and rhinovirus in the specimens from the ORF1a/b encodes overlapping replicase 1a and 1ab via the −1 ribo- young adults with acute respiratory infection (cough and body aches or somal frameshift at the nucleotide 12,424. The papain-like (PLpro) and chills or fever/feverishness) (Davis et al., 2018). Importantly, HCoV- 3C-like (3CLpro) proteases embedded within replicase 1a and 1ab NL63 is also associated with lower respiratory tract illnesses, such as process cis- and trans-cleavage activity to divide replicase 1a and 1ab pneumonia and bronchiolitis in young children and elderly (Huang ⁎ Corresponding author at: Department of Medical Laboratory Science and Biotechnology, China Medical University, 91 Hsueh-Shih Road, Taichung, 404, Taiwan. E-mail address: [email protected] (C.-W. Lin). 1 Co-first author. https://doi.org/10.1016/j.virusres.2019.197767 Received 20 June 2019; Received in revised form 20 September 2019; Accepted 23 September 2019 Available online 24 September 2019 0168-1702/ © 2019 Elsevier B.V. All rights reserved. J.-R. Weng, et al. Virus Research 273 (2019) 197767 et al., 2017). HCoV-NL63 infection is the high prevalence (8.4%) in 50 μg/ml penicillin, 50 μg/ml streptomycin, and 5% fetal bovine serum hospitalized patients with pneumonia in winter. Recently, a severe re- (FBS) at 37 ℃ in a 5％ CO2 incubator. LLC-MK2 cells were further used spiratory illness outbreak in a long-term care facility in Louisiana has to perform antiviral assays and examine antiviral mechanism. Human been reported to be associated with the HCoV-NL63 infection in winter airway Calu-3 cells were cultured in MEM supplemented 10% FBS and 2017 (Hand et al., 2018). Among 20 cases aged from 66 to 96, 6 pa- antibiotics mentioned above, maintained at the 80–90% confluent, and tients with pneumonia have to be hospitalized and 3 patients are dead. then also used to confirm the antiviral activity of indicated phenolic Moreover, screening children with acute undifferentiated febrile illness acid constituents. in rural Haiti indicates that HCoV-NL63 is identified in the blood samples from four patients aged from 3 to 10 years who have no re- 2.2. Preparation of Sambucusformosana Nakai stem ethanol extract spiratory symptom, but two cases have headache and the others exhibit influenza virus causing abdominal symptoms (Beau De Rochars et al., Dried stems of Sambucus Formosana Nakai (Supplemental Fig. 1A) 2017). Therefore, HCoV-NL63 is the notable pathogen as the etiology of were purchased from the medicinal herb pharmacy in Taichung, and mild and severe respiratory diseases, even acute undifferentiated febrile identified as described in Flora of Taiwan (Yang and Chiu, 1998). Dried illness. stem slices (Supplemental Fig. 1B) were soaked in 95% ethanol with the Sambucus FormosanaNakai, also known by Sambucus chinensis Lindl sonication for 2 h; the stem ethanol extract was filtered by Whatman and Sambucus javanica, is a species of elderberry, belongs to family No. 1 paper, then lyophilized in an IWAKI FDR-50 P freeze dryer, as Adoxaceae, and grows in subtropical and tropical Asian areas, including described in our previous study (Wang et al., 2012). The powder of Taiwan, China, Japan, Cambodia, India etc. (Lin and Tome, 1988; Yang Sambucus Formosana Nakai stem ethanol extract was kept in the sterile and Chiu, 1998; Hong et al., 2013). Sambucus FormosanaNakai is a bottle; the stock solution of 10 mg/ml in dimethyl sulfoxide was pre- traditional medicinal herb in Taiwan for reducing inflammation, en- pared and stored in −20 °C freezer and the test solutions of the stem hancing circulation, and treating rheumatoid and low back pain, extract were diluted by the media. The phenolic acid constituents such neuritis, dermatitis, and infection diseases (Yang and Chiu, 1998). The caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic chemical components of S.ambucus FormosanaNakai include sambuculin acid, were purchased from Sigma-Aldrich company. A, oleanolic acid, α-amyrin and β-amyrin, β-sitosterol, ursolic acid, pomolic acid, lupeol palmitate, glycyrrhetic acid, phenolic acid con- 2.3. Cell viability assay stituents, and flavonoids (Chen et al., 2001, 2019; Liao et al., 2006; Lin and Tome, 1988; Yang and Lin, 2004; Zhang et al., 2010). In addition, LLC-MK2 cells (3 × 104 cells/well) were cultured in the 96-well phenolic acid constituents of S.ambucus FormosanaNakai, including plates overnight, quintuplicate treated with Sambucus Formosana Nakai caffeic acid, caffeotannic acid, chlorogenic acid, coumaric acid, ferulic stem ethanol extract or its phenolic acid constituents (caffeic acid, acid, and gallic acid, have been identified as the members of the most chlorogenic acid, coumaric acid, ferulic acid, and gallic acid) for 2 days, important active components with anti-inflammatory, anti-tumor and and then incubated with 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5- anti-hepatotoxic activities (Chen et al., 2001; Liao et al., 2006; Yang diphenyltetrazolium bromide (MTT) for additional 4 h. After the cell and