Epigallocatechin gallate (EGCG), a green tea polyphenol possesses antioxidant, antibacterial, anticancer and antiviral properties. EGCG-Stearate (EGCG-S) is of interest for this study because of its stability and lipophilic properties. The chemical modification of EGCG-S increased its lipid solubility. Herpes simplex virus-1 (HSV-1), a member of the family Herpesviridae, and Alphaherpesvirinae subfamily is a leading cause of human viral diseases in the United States. In this study, 25 μM, 50 μM, 75 μM, and 100 μM of EGCG and EGCG-S were used to carry out cytotoxicity, cell viability and cell proliferation assays to determine the maximum non-cytotoxic concentrations on cultured A549 cells. The results suggested that 75 μM of EGCG and EGCG-S is the appropriate concentration to further study the effect on the infection of HSV-1 in A549 cells. Infectivity, antiviral, and inverted microscopy assays were performed to study the effects of EGCG and EGCG-S on HSV-1 infection. An antiviral assay was performed using luminescence and it indicated that EGCG-S treated HSV-1 showed up to 90% inhibition. Confocal microscopy images further supported the inhibitory effects of 75 μM EGCG-S on HSV-1 infection in A549 cells. The long-term goal of this research is to use EGCG-S as a possible novel topical therapeutic treatment to limit the spread of HSV-1 infections.
Herpes Simplex Virus-1 (HSV-1) is a member of the family Herpesviridae, and subfamily Alphaherpesvirinae. Herpesviruses are double-stranded, and enveloped DNA viruses that cause a wide range of diseases in humans and other animals [
Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, is the primary catechin obtained from leaves of the Camellia sinensis plant. EGCG has been previously demonstrated to have antiviral properties against several viruses including HIV, hepatitis B, hepatitis C, influenza, adenovirus, and Zika [
A549 human epithelial [America Type Culture Collection (ATCC), Manassas, VA, USA] cells were cultured in T25 flasks using F12-K media supplemented with 10% Fetal Bovine Serum (FBS) and 1 µg/mL gentamicin. Trypsin EDTA (0.25%) was used to subculture cells. The cells were maintained at 37˚C and 5% CO2.
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 [
EGCG (>90%) purchased from Pulimeidi Biotechnology Co., Ltd. (Hangzhou, China) and EGCG-S (US Patent 20120172423) modified by and purchased from Camellix, LLC, Augusta, GA, were dissolved in dimethyl sulfoxide (DMSO) and ethanol to prepare initial 5 mM stock concentrations. EGCG and EGCG-S were then diluted in F12-K media to desired concentrations of 12.5, 25, 50, 75, and 100 µM.
A549 cells were plated in 6 well plates, grown for 24 hours and treated with various concentrations (25, 50, and 75 µM) of EGCG and EGCG-S. After 1 hour the EGCG and EGCG-S were removed by aspiration and the cells were washed with phosphate buffered saline (PBS). Media was then added to the wells and cells were incubated at 37˚ under 5% CO2 and observed every 24 hours for a period of three days after treatment. Cells were examined, using an inverted microscope, at 400× magnification to observe morphological changes in the cells.
A549 cells were plated in 6 well plates and treated with appropriate concentrations of EGCG and EGCG-S for 1 hr. The viable cells were then stained and counted using trypan blue and hemocytometer. After 1 hr, the EGCG and EGCG-S were aspirated and the cells were washed with PBS. F12K media was then placed in each well and cells were incubated for 24 hrs. Cells, including controls of ethanol (ETOH) and DMSO, were trypsinized and harvested. Cells were then stained with trypan blue, which stains dead cells blue while live cells are not stained, and counted using a hemocytometer. Cell viability was determined by the proportion of viable cells to control levels at different treatments, and illustrated as relative cell viability with the controls as 100% viable.
Cell viability (%) = total viable cells (unstained)/total cells (stained and unstained) × 100
A549 cell suspensions (100 µL) were plated in separate wells in a 96 well plate and after 24 hours cells were treated with appropriate concentrations of EGCG and EGCG-S for one hour, then aspirated (as described in section 2.5). After 24 hours, 10 µL of WST-1 reagent (Roche Diagnostics, Indianapolis, IN, USA) was then added to the samples; the plate was gently rocked, then incubated at 37˚C under 5% CO2 for 30 minutes. The absorbance level for each well of the plate was measured at 450 nm in a 96 well plate reader. The assay was performed in triplicate.
A549 cells were plated in a 96 well plate and after 24 hours, 100 µL of virus was treated with 100 µL EGCG-S (final concentrations are 25, 50, 75, and 100 µM) for one hour. After treatment, cells were infected with treated and non-treated virus and incubated for one hour at 37˚C and 5% CO2. Any unabsorbed virus was aspirated and replaced with 100 µL of F12-K media. After 72 hours, 10 µL of ToxGlo reagent (Promega, Madison, WI, USA) was added to all wells containing samples (controls included 100 µL of 10% FBS-F12-K media both with and without the ToxGlo reagent) then incubated at 37˚ and 5% CO2 for 15 minutes. The plate was then read through a luminometer and relative light unit (RLU) values of each well were recorded. The assay was performed in triplicate.
The equation for calculating the percent of inhibition is shown below:
% inhibition = [(Cells + treated HSV1) − (Cells + HSV1)]/[Cells only − (Cells + HSV1)]
A549 cells were plated in 6 well plates and were infected with 200 µL of various dilutions of treated or non treated HSV-1 for one hour incubation, with intermittent rocking at 37˚C and 5% CO2. After one hour, unabsorbed virus was aspirated and 2.5 mL of media was added to each well and incubated at 37˚C and 5% CO2. Morphological changes were observed at day 3 post-infection.
A549 cells were grown on glass cover slips within 12 well plates and were infected with treated or non-treated virus for one hour. Twelve hours post infection, cells were stained with 300 µL of 300 nM DAPI (4,6-diamidino-2-phenylindole) stain for 5 minutes at 37˚C in the dark. Cells were then fixed with polyethylene glycol solution for 15 minutes at 20˚C. The glass cover slip containing cells were then glued to a slide using a drop of clear nail polish. Cells were then visualized and were examined under a Leica SP5 scanning confocal microscope under 10× or 63×/1.4 NA water Plan Apo objectives at Vassar College (Poughkeepsie, NY).
To study the effect of ECGC and EGCG-S on A549 human epithelium cells, different concentrations (25, 50 and 75 µM) of EGCG and EGCG-S were used. The morphology was observed at 24, 48 and 72 hours after the treatment as shown in
The cell viability was determined using the Trypan blue assay. After treatment with Trypan blue, cells were counted using a hemocytometer. The percent (%) of viability was calculated as explained in the Materials and Methods. The results are shown in
cell viability, with % viability of 98% and 100%, respectively, when compared with the control. For EGCG the % of viability ranges from 89% to 95%; for EGCG-S, the % of viability ranges from 83% to 87%. The cell proliferation assay using WST-1 was carried out to analyze the proliferation activity of cells. The results are shown in
The ToxGlo antiviral assay was used to measure cell viability in infected cells in response to EGCG-S treatment of the virus. The calculated percentage of inhibition ranged from ~80% at the highest concentration of EGCG-S while the maximum viral inhibition occurred at a concentration of 75 µM (>90%) (
The equation for calculating the percent of inhibition is given in Section 2.7 of the materials and methods.
A549 cells were plated in 6 well plates and were infected with treated and non treated HSV-1 for one hour. After an hour, unabsorbed virus was aspirated and media was added. Morphological changes were assessed at three days post-infection. The results are shown in
HSV-1, treated with 75 µM of EGCG-S, infected cells showed very similar cell morphology to the untreated control cells (c). This study suggests that 75 µM of EGCG-S was able to inhibit HSV-1 infection in cultured A549 cells thus the cells’ morphology remained similar to the morphology of the uninfected control.
HSV-1 used in this study contains the sequence for GFP fused to the sequence for the tegument protein, UL-46 [
indicates extensive HSV-1 infection. However, when HSV-1 is treated with 75 µM EGCG-S there is minimal GFP expression as shown in
There is a need to develop a treatment for herpes virus infections. Several natural compounds have been investigated as potential therapies. These natural products include eucalyptus extracts [
Herpes simplex virus infections continue to affect a large percentage of the human population worldwide. There is no cure for HSV-1 infections and there remains a need to identify effective and affordable therapies to reduce the incidence. The current treatment for herpes infections is acyclovir and its derivatives. Acyclovir is stable for oral and topical application; however, HSV resistance to nucleoside analogues has been reported due to mutations in viral thymidine kinase or polymerase [
Our results indicate that EGCG and EGCG-S can be safely applied to cultured A549 cells at concentrations up to 75 µM. Inhibition was measured visually as well as quantitatively. The ability of EGCG-S to inhibit HSV-1 infection was measured by a variety of assays including WST-1 cell proliferation assay, ToxGlo antiviral assay, inverted and confocal microscopy. The results of these assays demonstrate the inhibitory effects of treatment of HSV-1 virions with EGCG-S. EGCG has been reported to interact directly with HSV virions to inhibit attachment [
EGCG-S, a more stable and lipid soluble derivative of EGCG, does not affect cellular morphology; is not cytotoxic; and can inhibit the infection of HSV-1 in cultured cells. EGCG-S shows promise for use as a topical therapeutic treatment to limit the spread of HSV-1 infections.
This research was funded in part by the Science Honors Innovation Program (SHIP) at Montclair State University and by the Faculty Scholarship Program.
The authors declare no conflicts of interest.
SDA and LHL designed the study. SDA supervised SP in the laboratory. SP, SDA and LHL drafted the manuscript. SP conducted all experiments. All authors read and approved the final manuscript.
Patel, S.N., Adams, S.D. and Lee, L.H. (2018) Inhibition of Herpes Simplex Virus-1 by the Modified Green Tea Polyphenol EGCG-Stearate. Advances in Bioscience and Biotechnology, 9, 679-690. https://doi.org/10.4236/abb.2018.912046