Effect of black tea extract on herpes simplex virus-1 infection of cultured cells

Cantatore et al. BMC Complementary and Alternative Medicine 2013, 13:139 http://www.biomedcentral.com/1472-6882/13/139 RESEARCH ARTICLE Open Access Effect of black tea extract on herpes simplex virus-1 infection of cultured cells Anthony Cantatore, Sade D Randall, Daniel Traum and Sandra D Adams* Abstract Background: The purpose of this investigation was to determine if black tea extract (BTE), consisting primarily of flavanol compounds called theaflavins, could inhibit herpes simplex virus type-1 (HSV-1) infection in cultured A549 (human epithelial) and Vero cells. Methods: The effect of BTE both on A549 and Vero cultured cells and on HSV-1 was assessed by using phase contrast and fluorescent microscopy, and cell viability and proliferation assays. After establishing the maximum non-cytotoxic concentration of BTE, A549 and Vero cells and HSV-1 virions were treated with varying concentrations of BTE, respectively. A549 and Vero cells were infected with HSV-1 with green fluorescent protein (GFP) insert at the UL46 gene. The effect of infectivity was determined by viral DNA extraction followed by PCR, plaque assays, adsorption assays, and electrophoresis of PCR products. Results: BTE was not cytotoxic to A549 and Vero cells, as confirmed by cell viability and proliferation assays, in which BTE treated groups paralleled the positive control group. For both cell lines, plaque assays and fluorescent microscopy indicated an inverse relationship between BTE concentration (from 0.14 μM – 1.4 mM) and HSV-1 infectivity. Specifically, PCR and electrophoresis showed a reduction in the viral genome following treatment with BTE. In addition, there was a noticeable decrease in the amount of viral plaques for BTE treated samples in the adsorption assays. Conclusions: BTE consisting primarily of theaflavins is not cytotoxic and can reduce or block the production of infectious HSV-1 virions in cultured A549 and Vero cells, thus inhibiting the infectivity of the virus by interfering in the attachment, penetration and viral DNA replication of HSV-1 particles. These findings indicate that BTE enriched with theaflavins has the potential to be developed as a safe, therapeutic antiviral agent to prevent the spread of HSV-1. Keywords: Herpes simplex virus-1, Black tea extract, Theaflavins, Antiviral Background Herpes simplex virus type-1 (HSV-1) virions consist of an inner core with linear, double-stranded DNA that is enclosed in a capsid; an outer envelope containing various glycoproteins covers tegument proteins, which are exterior to the viral capsid [1,2]. The lytic infection cycle of HSV-1 begins with adsorption: when the virion first attaches to, then fuses with a host cell. Both attachment and penetration take place when viral glycoproteins bind to suitable receptors on the plasma membrane of the host cell. The presence of the * Correspondence: Department of Biology and Molecular Biology, Montclair State University, Montclair, NJ 07043, USA receptors determines the tropism of HSV-1 and places a limit on the types of cells to which it is capable of attaching, and thus, infecting [3-5]. Green, oolong, and black teas are produced from the same plant, Camellia sinensis. While the main type of flavanol in green tea extract is the catechin, that in BTE is the theaflavin, a dimer of different catechins, which includes: theaflavin (TF-1), theaflavin-3-monogallate (TF-2A), theaflavin-3'monogallate (TF-2B) and theaflavin-3,3'-digallate (TF-3) [6,7]. Since green tea is not fermented, it contains a relatively high amount of catechins as compared to black tea; fermentation causes the catechins to polymerize, which produces the theaflavins and thearubigens found in black tea, but absent in green tea [6]. © 2013 Cantatore et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cantatore et al. BMC Complementary and Alternative Medicine 2013, 13:139 http://www.biomedcentral.com/1472-6882/13/139 The benefits of tea are often attributed to its antioxidant properties, which in turn have been ascribed to catechins, since green tea extract has been found to have more antioxidant activity than black tea extract [7]. However, recent studies have shown that concentrated theaflavin extracts made from black tea can be just as effective as catechins; it is believed that the high number of hydroxyl (OH) groups of theaflavins, which have been shown to protect cells against oxidative damage, is responsible for its antioxidative properties [8,9]. Additionally, it has been found that theaflavins are capable of inhibiting certain types of cancer [10], as well as inhibiting viruses, including bovine rotavirus and bovine coronavirus [11], HIV-1 [12,13], and three different subtypes of influenza strains [14]. While it has been shown that HSV-1 can be inhibited by compounds in green tea extract and a variety of other polyphenolic compounds [15], the purpose of this study was to determine if black tea extract with a concentrated amount of theaflavins (≥ 80%) (BTE) could also inhibit HSV-1. Since theaflavins in BTE are composed of a dimer structure formed from catechin monomers found in green tea, which have been found to inhibit HSV-1 [15], it is reasonable to infer that theaflavins in BTE may also produce similar results based on structural similarities. Despite the fact that theaflavin molecules are larger than catechins, larger polyphenolic compounds such as tannins have been shown to inhibit other viruses [16,17], indicating that the size of the molecule may not necessarily be a factor required for viral inhibition. Rather, the large amount of hydroxyl groups on these polyphenolic compounds seem to be the one common structural component among these various, natural viral inhibitors; thus, BTE, with high concentrations of theaflavins, may be an effective inhibitor of HSV-1. Methods Cells Human epithelial (A549) cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured until confluent in 1X Ham's F-12K nutrient media, Kaighn's modification with 2 mM L-glutamine, supplemented to contain 10% fetal bovine serum (FBS) (Biowest, Miami, FL, USA) and 1 μg/mL gentamicin at 37°C and 5% CO2. Vero cells [ATCC (Manassas, VA)] were cultured until confluent in Dulbecco Modified Eagle Medium (DMEM) with 5% FBS and 1μg/mL gentamicin at 37°C and 5% CO2. HSV-1 UL-46 virus maintenance A recombinant strain of HSV-1, GHSV-UL46, which contains the sequence for green fluorescent protein (GFP) fused to the tegument protein pUL46, was used for all experiments [18] (ATCC, Manassas, VA, USA). Page 2 of 10 Passage of virus was performed in T-25 flasks and cells were allowed to reach complete cytopathic effect (CPE). The media was then collected, centrifuged, an (...truncated)