Research article
Antiviral activity of ginsenosides against coxsackievirus B3, enterovirus 71, and human rhinovirus 3
- Open Access funded by The Korean Society of Ginseng
- Under a Creative Commons license
Abstract
Background
Ginsenosides
are the major components responsible for the biochemical and
pharmacological actions of ginseng, and have been shown to have various
biological activities. In this study, we investigated the antiviral
activities of seven ginsenosides [protopanaxatriol (PT) type: Re, Rf,
and Rg2; protopanaxadiol (PD) type: Rb1, Rb2, Rc, and Rd)] against
coxsackievirus B3 (CVB3), enterovirus 71 (EV71), and human rhinovirus 3
(HRV3).
Methods
Assays of
antiviral activity and cytotoxicity were evaluated by the sulforhodamine
B method using the cytopathic effect (CPE) reduction assay.
Results
The
antiviral assays demonstrated that, of the seven ginsenosides, the
PT-type ginsenosides (Re, Rf, and Rg2) possess significant antiviral
activities against CVB3 and HRV3 at a concentration of 100 μg/mL. Among
the PT-type ginsenosides, only ginsenoside Rg2 showed significant
anti-EV71 activity with no cytotoxicity to cells at 100 μg/mL. The
PD-type ginsenosides (Rb1, Rb2, Rc, and Rd), by contrast, did not show
any significant antiviral activity against CVB3, EV71, and HRV3, and
exhibited cytotoxic effects to virus-infected cells. Notably, the
antiviral efficacies of PT-type ginsenosides were comparable to those of
ribavirin, a commonly used antiviral drug.
Conclusion
Collectively,
our findings suggest that the ginsenosides Re, Rf, and Rg2 have the
potential to be effective in the treatment of CVB3, EV71, and HRV3
infection.
Keywords
- antiviral activity;
- CVB3;
- EV71;
- ginsenosides;
- HRV3
1. Introduction
The Picornaviridae are currently divided into nine genera, three of which (Hepatoviruses, Rhinoviruses, and Enteroviruses) are causative agents of human diseases [1]. Enteroviruses
such as coxsackievirus, poliovirus, and echovirus are small,
nonenveloped viruses possessing a single-stranded RNA genome in positive
orientation that acts directly as mRNA in infected cells. Enteroviruses
are of high clinical relevance with coxsackievirus B3 (CVB3), which can
cause heart-muscle infection, being an important member. In addition,
Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth
disease and herpangina that can also cause severe neurological disease
including brainstem encephalitis and poliomyelitis-like paralysis [2], [3], [4] and [5]. Human rhinovirus (HRV) represents one of the most important etiological agents of the common cold [6].
Although HRV-induced upper respiratory illness is usually mild and
self-limiting, there is increasing evidence linking HRV infection to
more serious medical complications including asthma exacerbation [7].
To
date, no effective antiviral therapies have been approved for either
the prevention or treatment of diseases caused by viruses classified
within the Picornaviridae family, including CVB3, EV71, and HRV [8].
In this regard, many trials have been conducted to find antiviral
components from plants. Such trials have specifically targeted plants
with intrinsic defense mechanisms in the form of secondary metabolites
against a broad range of viral infections, in contrast to adaptive
immunity induced in mammals. Indeed, medicinal plants are gaining
popularity as suitable alternative sources of antiviral agents because
of their multiple targets, minor side effects, low potentials to cause
resistance, and low costs [9], [10], [11], [12] and [13].
Although several hundreds of plants with the potential to contain novel
antiviral agents have been studied, a number of potentially useful
medicinal plants still need to be evaluated and exploited for
therapeutic applications against the genetically and functionally
diverse virus families. Of these potential agents, we have focused on
ginsenosides, which are some of the major components of the ginseng
plant, Panax ginseng Meyer. The root of P. ginseng (Araliaceae) is the most well-known medicinal plant in the Asian region and is frequently used in traditional medicine [14]. Ginsenosides are triterpenoid glycosides containing dammarane [15],
and are generally divided into two groups: the protopanaxadiol (PD) and
protopanaxatriol (PT) ginsenoside groups. The sugar moieties in the PD
group including Rb1, Rb2, Rc, Rd, Rg3, and Rh3 are attached at the
3-position of dammarane-type triterpenes, whereas the sugar moieties in
the PT group including Re, Rf, Rg1, Rg2, and Rh1 are attached at the
6-position of dammarane-type triterpenes [16]. As the major components in ginseng, ginsenosides have various biological activities such as anticancer [17], antiaging [18] and [19], and antitumor activities [20]. Moreover, the antiviral activities of ginseng against influenza virus [15], norovirus [21], and HBV [22]
have recently been reported. Although a variety of pharmacological
activities associated with seven ginsenosides (PT group: Re, Rf, and
Rg2; PD group: Rb1, Rb2, Rc, and Rd) have been demonstrated, antiviral
activities especially against CVB3, EV71 and HRV3, which are
representative viruses of the picornaviridae and have drawn a great
attention in terms of potential therapeutics, have not been reported.
Therefore, in the current study, we investigated the antiviral
activities of seven ginsenosides against CVB3, EV71, and HRV3.
2. Materials and methods
2.1. Viruses, cell lines, and reagents
CVB3,
EV71, and HRV3 were supplied by Korea Research Institute Bioscience and
Biotechnology, Ochang-eup, South Korea. A human cervix epithelial cell
line (HeLa, CCL-2) and African green monkey kidney cells (Vero, CCL-81)
were purchased from the American Type Culture Collection (Manassas, VA,
USA). HeLa and Vero cells were maintained in minimal essential medium
supplemented with 10% fetal bovine serum and 0.01%
antibiotic–antimycotic solution. Antibiotic–antimycotic solution,
trypsin–EDTA, fetal bovine serum and minimal essential medium were
supplied by Gibco BRL (Grand Island, NY, USA). Tissue culture plates
were purchased from Falcon (BD Biosciences, Franklin Lakes, NJ, USA).
Ribavirin and sulforhodamine B (SRB) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). The seven ginsenosides were obtained from Dr. Bae L
(Elohim, Co., Daejeon, South Korea). Stock solutions (100 mg/mL) of the
antiviral compounds were dissolved in dimethyl sulfoxide (DMSO) and
were subsequently diluted in the culture medium. The final DMSO
concentration in the culture medium did not exceed 0.1%, which was found
to have no visible toxic effect on the cells. As a negative control,
0.1% DMSO was also added to all no-drug control samples.
2.2. SRB assays of antiviral activity
Assays
of antiviral activity and cytotoxicity were evaluated by the SRB method
using cytopathic effect (CPE) reduction recently reported [23]. Briefly, 1 day prior to infection, Vero cells were seeded onto a 96-well culture plate at a concentration of 2 × 104 cells/well.
The following day, the culture medium was removed and cells were washed
with phosphate-buffered saline (PBS). The infectivity of each virus was
determined by the SRB method monitoring CPE, allowing for the
percentage of cell viability to be determined. Based on the mammalian
cell viability determined for each virus, 0.09 mL of diluted virus
suspension of CVB3 or EV71 containing CCID50 (50% cell
culture infective dose) of virus stock was added to mammalian cells.
This dose was selected to produce the appropriate CPEs 48 hours after
infection. For compound treatments, 0.01 mL of the medium containing the
selected concentration of compound was added to the cells. The
antiviral activity of each test material was determined using a 10-fold
diluted concentration range of 0.1–100 μg/mL. Four wells were used as
virus controls (virus-infected, nondrug-treated cells), whereas four
wells were used as cell controls (noninfected, nondrug-treated cells).
Culture plates were incubated at 37°C in 5% CO2 for 48 h.
After washing once with PBS, 100 μL of cold (−20°C) 70% (v/v) acetone
was added to each well and left for 30 min at −20°C. The acetone was
removed from cells, after which 96-well plates were left to dry in oven
at 60°C for 30 min. Then, 100 μL of 0.4% (w/v) SRB in 1% acetic acid
(v/v) was added to each well and incubated at room temperature for
30 min. Unbound SRB was removed by washing the plates five times with 1%
acetic acid (v/v), and the plates were then left to dry in an oven.
After drying for 1 day, cell morphology was assessed under a microscope
at 4 × 10 magnification (AXIOVERT10; Zeiss, Göttingen, Deutschland) and
images were acquired. Fixed SRB in wells was solubilized with 100 μL of
unbuffered Tris-base solution (10 mM), and plates were incubated at room
temperature for 30 min. Absorbance in each well was read at 540 nm
using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA,
USA) and a reference absorbance of 620 nm. The antiviral activity of
each test compound in CVB3- or EV71-infected cells was calculated as a
percentage of the corresponding untreated control.
2.3. Cell Titer-Glo assays of antiviral activity
The
antiviral activity of seven ginsenosides against HRV3 was determined
using a Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega,
Madison, Wisconsin, USA). The Cell Titer-Glo Reagent induces cell lysis
and the generation of luminescence proportional to the amount of ATP
present in cells. The resulting luminescence intensity is measured using
a luminometer (Molecular Devices) according to the manufacturer's
instructions. Briefly, HeLa cells were seeded onto a 96-well culture
plate, after which 0.09 mL of diluted HRV3 suspension containing CCID50 of the virus stock, and 0.01 mL culture medium supplemented with 20 mM MgCl2
and the appropriate concentration of ginsenosides, was added to the
cells. The antiviral activity of each test material was determined using
a concentration series of 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL.
Culture plates were incubated at 37°C in 5% CO2. After 48 h,
100 μL of Cell Titer-Glo reagent was added to each well, and the plate
was incubated at room temperature for 10 min. The resulting luminescence
was measured and the percentage cell viability was calculated as
described for the antiviral activity assays. Cell morphology was
assessed as described for the SRB assay.
2.4. Cytotoxicity
To measure cytotoxicity, cells were seeded onto a 96-well culture plate at a density of 2 × 104
cells/well. The following day, the culture medium containing serially
diluted compounds was added to the cells and incubated for 48 h, after
which the culture medium was removed and cells were washed with PBS. The
next step was conducted as described above for the antiviral activity
assay. To calculate the CC50 values, the data were expressed as percentages relative to controls, and CC50 values were obtained from the resulting dose–response curves.
2.5. Statistical analyses
Differences
across more than three groups were analyzed using one-way analysis of
variance (Graphpad PRISM, version 5.01, San Diego, CA, USA). All results
were expressed as mean ± standard deviation. Significant differences in
direct comparisons were determined using a Tukey's post hoc test. Differences with p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.
3. Results
3.1. Antiviral activity of ginsenosides against CVB3
The
antiviral activities of ginsenosides against CVB3 were assessed using
the SRB method, which monitors the alteration of CPE induced by virus
infection. As a positive control, ribavirin, a commonly used antiviral
drug, was included. Of the seven ginsenosides tested, ginsenosides Re,
Rf, and Rg2, which are classified as PT-type ginsenosides, significantly
inhibited CVB3-induced CPE, and increased the cell viability of Vero
cells (Fig. 1).
CVB3 infection induced approximately 60% cell death in Vero cells (40%
of cell viability), and the treatment of cells with 100 μg/mL of Re, Rf,
and Rg2 increased the cell viability to 75%, 60%, and 50%,
respectively. Furthermore, 10 μg/mL of ginsenosides Re and Rg2 also
significantly reduced the CPE of CVB3 infection in Vero cells, albeit a
weaker protective effect than that of ribavirin at the same
concentration. By contrast, the PD-type ginsenosides Rb1, Rb2, Rc, and
Rd did not exhibit any antiviral activity against CVB3, and 100 μg/mL of
Rd, Rc, and Rb2 even significantly increased CVB3 infection-induced
cytotoxicity (Fig. 1).
