Broad-spectrum antiviral properties of andrographolide

Abstract

Andrographolide, a diterpenoid, is known for its anti-inflammatory effects. It can be isolated from various plants of the genus Andrographis, commonly known as ‘creat’. This purified compound has been tested for its anti-inflammatory effects in various stressful conditions, such as ischemia, pyrogenesis, arthritis, hepatic or neural toxicity, carcinoma, and oxidative stress, Apart from its anti-inflammatory effects, andrographolide also exhibits immunomodulatory effects by effectively enhancing cytotoxic T cells, natural killer (NK) cells, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC). All these properties of andrographolide form the foundation for the use of this miraculous compound to restrain virus replication and virus-induced pathogenesis. The present article covers antiviral properties of andrographolide in variety of viral infections, with the hope of developing of a new highly potent antiviral drug with multiple effects.

Full text

1 23
Archives of Virology
Official Journal of the Virology
Division of the International Union of
Microbiological Societies
ISSN 0304-8608
Arch Virol
DOI 10.1007/s00705-016-3166-3
Broad-spectrum antiviral properties of
andrographolide
Swati Gupta, K.P.Mishra & Lilly Ganju

1 23
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REVIEW
Broad-spectrum antiviral properties of andrographolide
Swati Gupta
1
•K. P. Mishra
1
•Lilly Ganju
1
Received: 6 June 2016 / Accepted: 20 October 2016
ÓSpringer-Verlag Wien 2016
Abstract Andrographolide, a diterpenoid, is known for its
anti-inflammatory effects. It can be isolated from various
plants of the genus Andrographis, commonly known as
‘creat’. This purified compound has been tested for its anti-
inflammatory effects in various stressful conditions, such
as ischemia, pyrogenesis, arthritis, hepatic or neural toxi-
city, carcinoma, and oxidative stress, Apart from its anti-
inflammatory effects, andrographolide also exhibits
immunomodulatory effects by effectively enhancing cyto-
toxic T cells, natural killer (NK) cells, phagocytosis, and
antibody-dependent cell-mediated cytotoxicity (ADCC).
All these properties of andrographolide form the founda-
tion for the use of this miraculous compound to restrain
virus replication and virus-induced pathogenesis. The
present article covers antiviral properties of andro-
grapholide in variety of viral infections, with the hope of
developing of a new highly potent antiviral drug with
multiple effects.
Abbreviations
NK cells Natural killer cells
T
regs
Regulatory T cells
CTLs Cytotoxic T lymphocytes
IAV Influenza A virus
NS Non-structural
HBV Hepatitis B virus
NC Nucleocapsid
DCs Dendritic cells
HCV Hepatitis C virus
HSV Herpes simplex virus
EBV Epstein-Barr virus
HPV Human papillomavirus
HIV Human immunodeficiency virus
AIDS Acquired immunodeficiency syndrome
gp Glycoprotein
CNS Central nervous system
CHIKV Chikungunya virus
NCR Non-coding region
HBsAg Hepatitis B surface antigen
HBeAg Hepatitis B envelope antigen
14-DDA 14-Deoxy-11,12-didehydroandrographolide
DAD 14-Deoxyandrographolide
IPAD 3, 19-Isopropylideneandrographolide
ACV Acyclovir
HSV-1DR HSV-1 drug-resistant strain
CPE Cytopathic effect
RIG-1 Retinoic acid inducible gene 1
RLRs RIG-1-like receptors
HO-1 Haeme oxygenase 1
DASM Dehydroandrographolide succinic acid
monoester
BAL Bronchoalveolar
ADCC Antibody-dependent cell-mediated
cytotoxicity
Introduction
Viruses are infectious agents that infect host cells to
increase their progeny. In some cases, viral infection
results in lysis of the host cell leading to the release of
millions of copies of the virus. Viruses exploit the host cell
&K. P. Mishra
kpmpgi@rediffmail.com
1
Immunomodulation Laboratory, Defence Institute of
Physiology and Allied Sciences, Lucknow Road, Timarpur,
Delhi 110054, India
123
Arch Virol
DOI 10.1007/s00705-016-3166-3
Author's personal copy

by using their replication machinery and also affect the
energy balance required for the survival of the cell.
Imbalanced energy production and energy expenditure
results in apoptosis. Some viruses not only survive by
successfully evading the host immune system but are also
able to suppress it, causing various detrimental infectious
and opportunistic diseases. Chronic infection can cause
altered expression of housekeeping genes and proteins of
the cell, thus transforming it into a cancerous cell or
hyperactivating the immune system, thereby initiating the
destruction of host cells in order to kill virus-infected cells.
This state of activation triggers an autoimmune response
leading to destruction of host cells.
Some antiviral drugs stimulate the host immune system
to attack the virus, providing protection against a variety of
viral infections. These drugs are considered better than
virus-specific antiviral drugs, which act on entry, release,
replication, integration or other stages of the virus life
cycle. Their nonspecific mechanism of action can kill other
microorganisms and parasites, which is another advantage
of using immune-stimulating antiviral drugs.
Andrographolide is a drug that has antiviral (Table 1)
[1], antimicrobial [2–4] and anti-parasitic effects [5–7]. It
is a labdane diterpenoid (Fig. 1) that can be purified from
the aerial parts of various plants of the genus Andrographis
(family Acanthaceae), which grow at different elevations.
The bitter taste of the bioactive component of Andro-
graphis paniculata gave this plant the title of ‘‘King of
Bitter’’. A. paniculata is found in India, Sri Lanka, China,
Malaysia, Thailand, and Japan at an elevation of up to
1000 m [8]. It is an annual herb about 30-110 cm tall, with
white flowers with purple spots on the petals. Andro-
grapholide is soluble in organic solvents such as ethanol,
chloroform, ether, acetone, and dimethyl sulfoxide, and can
be extracted from A. paniculata,A. alata, and A. lineate
[9].
The anti-inflammatory effects of andrographolide can
efficiently neutralize cell-lysis-induced inflammation.
Andrographolide-mediated suppression of NF-jB and NO
contributes to its anti-inflammatory properties [10–13]. The
differential apoptosis of cancer cells can be induced by
andrographolide, which can inhibit virus-induced carcino-
genesis. Its pro-apoptotic role in hamster buccal pouch
carcinoma and human colorectal carcinoma has been
reported by Shanmugam et al. and Lin et al., respectively
[14,15]. Andrographolide induced apoptosis in human
cancer cell line [16] by activation of caspase 8, caspase-8-
dependent cleavage of Bid, and conformational changes in
Bax has been demonstrated by Zhou et al. [17]. Mito-
chondria-mediated apoptosis in lymphoma cells induced by
andrographolide has been reported by Yang et al. [18].
Further inhibitory effects of andrographolide on migration,
invasion and matrix metalloproteinase expression has also
been described in rheumatoid arthritis, angiogenesis, breast
cancer, human colorectal carcinoma and human small-cell
lung carcinoma [19–23]. In contrast to the role of andro-
grapholide as a pro-apoptotic agent, it can induce prolif-
eration of cytotoxic T lymphocytes (CTLs) both in vitro
and in vivo [24], which can kill virus-infected host cells.
Andrographolide plays a role in mitigation of autoimmune
responses. Iruretagoyena et al. reported the reduction of
experimental autoimmune encephalomyelitis by interfering
with T-cell activation [25]. Andrographolide also possesses
cytoprotective properties against various oxidative stresses.
It has a neuroprotective effect in permanently cerebral
ischaemic rats [26] and against nicotine–induced oxidative
stress in the brains of male Wistar rats [27]. It also has
hepatoprotective properties against carbon-tetrachloride-
induced oxidative damage in rats [28], ethanol-induced
hepatorenal toxicity in mice [29], and concanavalin-A-in-
duced liver injury [30]. Therefore, andrographolide
nanoparticles have also been engineered to aid in rapid
recovery from liver toxicity [31].
Andrographolide also modulates the host immune
response. Injection of killed Salmonella vaccine in mice
and subsequently feeding them with different quantities of
andrographolide resulted in a significant increase of S.
typhimurium-specific IgG antibodies [32]. Cyclophos-
phamide-induced delayed-type hypersensitivity was
reversed by a mixture of various purified labdane diterpe-
nes of A. paniculata [33]. Andrographolide also reduces
IL-2 production in T cells by interfering with nuclear factor
of activated T cells (NFAT) activation and ERK-1 and
ERK-5 phosphorylation [34]. Sheeja et al. reported that
CTL and NK cell activity were upregulated by andro-
grapholide treatment [35]. Peng et al. observed an increase
in phagocytic activity along with induction of the cytokines
IFN-a, IFN-c, and TNF-ain peripheral blood mononuclear
cells (PBMCs) [36].
The properties of andrographolide, such as its ability to
induce apoptosis of cancer cells and inhibition of DTH, its
anti-oxidative and cytoprotective effect, and its ability to
enhance CTLs and NK cell activation makes it a potent
antiviral agent. In the present review, we discuss the broad-
spectrum antiviral properties of andrographolide against
different viruses, which are summarized in Figure 2.
Influenza A virus (IAV)
Influenza A virus (IAV) is a negative-sense, single-strand
RNA virus belonging to the family Orthomyxoviridae and
genus Influenzavirus A. It is a spherical virus that is
80-120 nm in diameter with an external layer of spike-like
projections. IAV is a causative agent of respiratory infec-
tion in humans, and virus replication takes place in
epithelial cells of the upper and lower respiratory tract.
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Table 1 Antiviral effects of andrographolide on different viruses
Virus Genome type Cells affected Associated complications in humans In vivo or
in vitro studies
Dose of
andrographolide
used
Effect of andrographolide Ref.
Influenza A virus Negative-
sense
single-
stranded
RNA
Epithelial cells of the
upper and lower
respiratory tract
Respiratory infection including
pneumonia, sinus infections, asthma,
etc.
BALB/c mice
and MDCK
cells
4000 to 125 mg/
kg/d
1. Inhibits H9N2, H5N1 and H1N1
virus both in vitro and in vivo
[41]
DCs and
macrophages
250 lg/ml 2. Inhibits the H1N1-induced RIG-1-
like receptor signaling pathway
[44]
MDCK cells 200 to 3277.4
lg/ml
3. Inhibits H3N2 virus replication [45]
Hepatitis B virus Partially
double-
stranded
relaxed
circular
DNA
Hepatocytes, DCs,
T
regs
and NK cells
Liver cirrhosis, fibrosis and hepatocellular
carcinomas
HepG 2.2.15
cells
54.1 to 200 lM Inhibits HBV DNA replication [55]
Hepatitis C virus Positive-sense
single-
stranded
RNA
Hepatocytes, PBMCs,
especially B cells
Chronic hepatitis, liver cirrhosis, and
hepatocellular carcinoma and
extrahepatic infection, resulting in B cell
non-Hodgkin’s lymphoma
Huh 7 cells 1 to 10 lM Suppresses HCV genome replication
by promoting IFNaresponse, viral
HO-1 gene activity and inhibiting
viral NS3/4A protease activity
[60]
Molecular
docking
study
- Inhibits HCV NS3/4A protease and
its drug-resistant mutants
[61]
Herpes simplex virus
1
Double-
stranded
DNA virus
Skin and mucosal
epithelial cells
Blister formation at skin and mucosal
membrane of mouth, lips, genitals and
oesophagus and conjunctivitis, keratitis,
iridocyclitis and acute retinal necrosis in
the ocular region
Vero cells 8- 89 lg/ml 1. Reduces HSV-1-induced plaque
formation
2. Inhibits HSV-1 DNA replication
and gp C and D expression
[66]
Vero cells 16.28 -76.3 lM Inhibits HSV-1 entry into the cell [67]
Vero cells 20.50 lM Andrographolide analogue, 3,
19-isopropylideneandrographolide
inhibits HSV-1 wild-type and drug
resistant strains’ DNA and protein
synthesis
[68]
Epstein-Barr virus Linear
double-
stranded
DNA virus
B cells and epithelial
cells of salivary
gland and also T
cells, NK cells and
smooth muscle cells
Nasopharyngeal carcinoma, Burkitt’s
Lymphoma, Hodgkin’s Lymphoma,
gastric carcinoma, multiple sclerosis and
lymphomatoid granulomatosis
P3HR1 cells 1- 10 lg/ml Inhibits two viral immediate early
genes, resulting in inhibition of
viral lytic protein expression
[73]
Human
papillomavirus
Double-
stranded
circular
DNA
Basal epithelial cells Genital warts and cancer in cervical,
vulvar, vaginal, penile, anal, and
oropharyngeal region
CaSki cells 9- 152.34 lM Inhibits E6 oncogenic envelope gp
and restores tumor suppressor p53
protein
[76]
Andrographolide as an antiviral agent
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Patients with IAV release aerosol particles containing the
virus into the environment, which leads to the spread of the
virus to a new host [37]. IAV infects variety of animals,
including waterfowl as their original reservoir, whales,
seals, cats, dogs, swine, poultry, and humans. The genome
of IAVs consists of eight segments [38]. Segment 1-6 of
the IAV genome code for single proteins, i.e., polymerase
PB2, polymerase PB1, polymerase PA, haemagglutinin
(HA), nucleoprotein and neuraminidase (NA), respec-
tively. Segments 7 and 8 have overlapping reading frames
encoding matrix proteins (MI and M2) and nonstructural
(NS) proteins (NS1 and NS2), respectively. IAV has two
surface glycoproteins (gp), namely HA, a trimer consisting
of three identical subunits, and NA, a tetrameric spike [39].
HA binds to a sialic acid receptor to allow infection of the
host, whereas NA cleaves the receptor to allow the release
of the virus. Human bronchoalveolar (BAL) fluid exhibits
innate immune defense activity against influenza virus.
Resistance to BAL fluid of a drug-resistant H1N1 virus due
to its NA gene activity has been shown to result in
increased viral fitness [40]. Therefore, more-efficient
antiviral drugs are required to inhibit viral gene activity,
which should slow down the evolution of drug-resistant
forms.
Andrographolide has been proposed to be a very
effective drug against IAV. Chen et al. showed that
andrographolide and its various derivatives inhibit H9N2,
H5N1 and H1N1 strains of influenza virus, both in vitro
and in vivo [41]. Andrographolide has also been screened
using the Lipinski rule, which evaluates if compounds with
biological activity can be used as drugs in humans. Raja
et al. screened andrographolide using the Lipinski rule and
found that andrographolide, with a molecular weight
Fig. 1 Structure of andrographolide
Table 1 continued
Virus Genome type Cells affected Associated complications in humans In vivo or
in vitro studies
Dose of
andrographolide
used
Effect of andrographolide Ref.
Human
immunodeficiency
virus
Two identical
copies of
single-
stranded
positive-
sense RNA
Macrophages,
monocytes, DCs and
microglial cells and
CD4
?
T cells
AIDS and opportunistic infections H9 cells 50- 200 lg/ml 1. Increases CD4
?
T cell count [79]
MT2 cells 5 to 100 mg/mL 2. Reduces p24 antigen levels [80]
Chikungunya virus Single-
stranded
positive-
sense RNA
Epithelial, endothelial,
fibroblasts,
monocytes and
macrophage cells
Fever, headache, rashes, myalgia, and
polyarthralgia
HepG2 cells 1 to 100 lM Reduces viral RNA copy number
and inhibits viral protein
expression
[83]
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350.455 (must be B500) has three hydrogen bond donors
(must be B5) and five hydrogen bond acceptors (must be
B10). In addition, a molecular docking study revealed that
andrographolide forms five hydrogen bonds with HA and
three H bonds with NA with binding energy values of -6.48
and -7.04 kcal/mol, respectively, signifying the interaction
of andrographolide with virus proteins in regulating various
biological processes [42]. Host innate immune factors such
as retinoic acid inducible gene-1 (RIG-1)-like receptors
(RLRs) are involved in detection of RNA viruses inside the
cytoplasm. The RLR family includes RIG-1, MDA5, and
LGP2, which, on sensing RNA viruses, induce the initia-
tion and modulation of antiviral immunity of the host [43].
Infection with H1N1 leads to the activation of the RLR-
dependent signaling pathway. Andrographolide inhibits the
H1N1-induced RIG-1-like receptor signaling pathway in
human bronchial epithelial cells, indicating inhibition of
virus-induced activation of the RLR pathway, leading to
amelioration of H1N1-virus-induced cell mortality [44].
Yuan et al synthesized 18 new semi-synthetic andro-
grapholide analogues and bio-assayed their anti-influenza
A virus (H3N2) activity in vitro. Among the compounds
synthesized, benzyl amino derivative 38 showed the
highest potency and was 1.5 times more efficient than
lianbizhi, an andrographolide analogue used in Chinese
medicines [45]. Moreover, the effectiveness of A. panicu-
lata extract SHA-10 on patients suffering from the com-
mon cold was evaluated by Caceres et al. by visual
analogue scale measurement [46]. Their study concluded
that SHA-10 dried extract (1200 mg/day) effectively
reduced the prevalence and intensity of uncomplicated
common cold symptoms at day two of treatment.
Hepatitis B virus
Hepatitis B virus (HBV), a DNA virus, belongs to the
family Hepadnaviridae and genus Orthohepadnavirus.
Hepatitis B is an infectious disease caused by HBV
infection, which affects hepatocytes, leading to cirrhosis,
fibrosis and hepatocellular carcinomas. HBV is about
42 nm in diameter, consisting of an outer lipoprotein coat
and the hepatitis B surface antigen (HBsAg). The HBV
genome is 3.2 kb long and is a partially double-stranded,
relaxed circular DNA packaged in a nucleocapsid (NC).
HBsAg can either exist as viral-particle-bound protein or as
a free noninfectious protein. In utero viral exposure to fetal
Fig. 2 Effects of
andrographolide on the immune
system and virus entry and
propagation
Andrographolide as an antiviral agent
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immune cells results in a symbiotic relationship between
HBV and its host, which might have lead to the wide
distribution of HBV to large part of the human population
[47]. Vaccines against HBV contain HBsAg to generate
neutralizing antibodies providing long-term protection
[48].
The transmission of HBV mostly takes place percuta-
neously, sexually, perinatally and through blood transfu-
sion, but patterns of HBV transmission vary throughout the
world. HBV can infect humans and non-human primates of
the families Hominidae (chimpanzees, gorillas, and oran-
gutans) and Hylobatidae (gibbons) [49]. HBV infection
activates CD4 and CD8 T-cell and B-cell responses.
However, the presence of immunosuppressive regulatory T
cells (T
regs
) contributes to an inadequate immune response
against HBV, causing a chronic infection [50]. The
immune cells and pathways targeted by HBV include
dendritic cells (DCs), T
regs
, NK cells, and the interferon
(IFN) pathway, respectively [51].
A number of chemical or plant-derived compounds have
been screened and approved for HBV treatment [52,53].
The side effects of the currently approved drugs and
resistance developed by the virus has forced a further
search for anti-HBV agents [54]. Chen et al. synthesized 48
derivatives of dehydroandrographolide and andro-
grapholide and evaluated their anti-HBV activity. They
reported that andrographolide and some of its derivates not
only inhibit HBsAg and hepatitis B envelope antigen
(HBeAg) secretion but also exhibit anti-HBV effects by
inhibiting HBV DNA replication [55]. They also investi-
gated the relationship of the structure and anti-HBV
activity of andrographolide and its derivatives.
Hepatitis C virus
Hepatitis C virus (HCV) is a single-strand, positive-
sense RNA virus belonging to the family Flaviviridae,
genus Hepacivirus. HCV mostly infects hepatocytes, and
recently,HCVwasalsofoundtoinfectperipheral
mononuclear lymphocytes, especially B cells expressing
CD81 molecules [56]. HCV infection of the liver causes
chronic hepatitis, liver cirrhosis, and hepatocellular car-
cinoma and extrahepatic infection results in B-cell non-
Hodgkin lymphoma. HCV is about 55-65 nm in diameter
and contains a genome of approximately 9.6 kb. The
HCV genome encodes 10 viral proteins. The viral
structural proteins include the capsid and envelope gly-
coproteins E1 and E2. In the polyprotein precursor, the
NS protein p7 separates the structural proteins from the
other NS proteins and has viroporin activity. Other NS
proteins include NS2, NS3, NS4A, NS4B, NS5A and
NS5B, which are involved in viral replication and
polyprotein processing.
Host-pathogen interactions provide the stimulation to
the host immune system that is required for virus clearance.
HCV infection can activate the production of IFNs, which
restrict virus propagation. However, HCV has developed
strategies to counter host antiviral immune responses. In
order to inhibit IFN production, HCV NS4B suppresses
stimulator of IFN gene [57]. Immune cells such as CTLs
can also restrict virus multiplication inside the host.
Although CTLs can limit HCV replication, these cells are
also responsible for liver damage, leading to chronic HCV
infection [58]. Therefore, antiviral drugs with bivalent
action are required that can limit virus replication and can
also stimulate a host antiviral immune response.
Many anti-HCV drugs have been designed and tested for
their viral polymerase, NS3/4A protease and cyclophilin
inhibition activities [59]. The ability of HCV to develop
resistance very rapidly has led to a further rising demand
for new anti-HCV drugs. Lee et al. combined andro-
grapholide with IFN-a, telaprevir (HCV NS3/4A protease
inhibitor), and PSI-7977 (an inhibitor targeting HCV NS5B
polymerase) in an attempt to develop an effective antiviral
drug [60]. They reported significant synergistic effects of
andrographolide with these drugs. Andrographolide
increased haeme oxygenase-1 (HO-1) and as a result, liver
biliverdin increased, which suppressed HCV replication by
enhancing the IFN response and inhibited NS3/4A protease
activity. Andrographolide also activated p38 MAPK
phosphorylation, which led to the activation of nuclear
factor erythroid 2-related factor 2 (Nrf2)- mediated HO-1
expression, which was also linked to anti-HCV activity.
Chandramohan et al. also predicted andrographolide to be a
potent inhibitor of wild-type HCV NS3/4A protease and its
drug-resistant mutants R155K and D168A through
molecular docking studies [61]. Molecular docking simu-
lations also showed andrographolide to have good target-
protein binding ability and to maintain strong bonds,
causing little disturbance of the protein backbone structure.
The multiple modes of action of andrographolide in
restraining HCV activity shows that this compound is a
promising candidate for advanced research.
Herpes simplex virus 1 (HSV-1)
Herpes simplex virus 1 belongs to the family Herpesviri-
dae, genus Simplexvirus. It is an enveloped, double-stran-
ded DNA virus with a genome size of 152 kb and a
diameter of approximately 200 nm. HSV has four distinct
structural layers, which include an outermost envelope, a
matrix or tegument, an inner capsid of icosahedral sym-
metry, and the DNA core. HSV-1 DNA consists of two
unequal regions, i.e., a long (L) segment and a short
(S) segment and encodes over 80 proteins. The HSV-1
genes are divided into immediate early genes, early genes,
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and late genes based on their order of transcription and
translation. HSV-1 infects skin and mucosal epithelial cells
and undergoes a lytic cycle, whereas in neuronal cells, it
becomes latent. HSV-1 infection leads to blister formation
on skin and mucosal membranes of the mouth, lips, geni-
tals and oesophagus. In the ocular region, HSV-1 infection
leads to conjunctivitis, keratitis, iridocyclitis, and acute
retinal necrosis [62].
The host defence against HSV infection includes both
innate and adaptive immune response. However, HSV-in-
fection inhibits phenotypic and functional maturation of
DCs subsequently resulting in reduced function of HSV
specific CD8
?
T cells [63]. The United States has licensed
drugs for the treatment of HSV infection, including acy-
clovir (ACV) for neonatal HSV disease, herpes simplex
encephalitis, mucocutaneous and viscerally disseminated
herpes infection. However, resistance to ACV can develop
due to mutations in the viral thymidine kinase. Another
FDA-approved drug for the treatment for acute herpes
zoster is famciclovir. However, similar to ACV, resistance
to famciclovir arises due to mutations in the viral thymi-
dine kinase gene [64]. To deal with the evolution of drug-
resistant forms, more antiviral drugs are required.
Andrographolide and other derivatives from A. panicu-
lata, such as neoandrographolide and 14-deoxy-11,12-
didehydroandrographolide (14-DDA), have been shown to
reduce the number of plaques formed by HSV-1 in Vero
cells [65]. Seubsasana et al. also showed that andro-
grapholide and 14-deoxyandrographolide (DAD) isolated
from A. paniculata and 3, 19-isopropylideneandro-
grapholide (IPAD), a semi-synthetic compound of andro-
grapholide inhibited HSV entry less than 50% [66].
However, post-infection administration of IPAD resulted in
100% inhibition of HSV infection, and this drug exhibited
anti-replication activity and reduced the expression of gp C
and gp D at all intervals of treatment. This study also
showed that IPAD did not have an inhibitory effect on the
immediate early step of viral replication, since inhibition
started at 4 h postinfection.
Aromdee et al. modified three free hydroxyls at C-3,
C-14 and C-19 of andrographolide, 14-DDA, DAD and
eight semisynthetic analogues and explored their anti-HSV
activity [67]. Their results confirmed that these three
hydroxyl moieties play an important role in the anti-HSV-1
activity of andrographolide. They also found that 14-acetyl
analogues block viral entry, whereas IPAD exerts post-
infection anti-HSV-1 activity. Priengprom et al. reported
the synergistic effects of the andrographolide analogue
IPAD with ACV against infection with wild-type HSV
(HSV-1 strain KOS and HSV-2 clinical isolate) and an
HSV-1 drug-resistant strain (HSV-1DR). They reported
that a non-cytotoxic concentration of IPAD (20.5 lM)
completely inhibited the cytopathic effect (CPE) caused by
wild-type HSV and HSV-1DR. A combination of IPAD
and ACV synergistically inhibited CPE, viral DNA repli-
cation, and protein synthesis in cells infected with wild-
type HSV and HSV-1DR [68].
Epstein-Barr virus (EBV)
Epstein-Barr virus belongs to the family Herpesviridae
and genus Lymphocryptovirus. EBV is a 120- to 150-nm
linear double-stranded DNA virus that produces virions
with icosahedral symmetry. The EBV NC is about
100-200 nm in diameter and contains a genome of
172 kb. EBV infects B cells and epithelial cells of
salivary glands, and under some circumstances it can
also infect T cells, NK cells and smooth muscle cells.
Transmission of virus takes place through blood, saliva
and genital secretions. It is a major cause of ‘‘kissing
disease’’, or mononucleosis, as the virus is mostly
transmitted through the saliva of an infected person.
EBV can be transmitted within epithelial cells through
contact by the formation of cell-in-cell structures
[69,70]. In epithelial cells, the virus undergoes repli-
cation and finally completes the lytic cycle, resulting in
transmission of virus to B cells.
EBV infection causes fatigue, rash, sore throat, swollen
glands, weakness, and other symptoms. Along with acute
infectious mononucleosis, EBV also causes nasopharyn-
geal carcinoma, Burkitt’s lymphoma, Hodgkin’s lym-
phoma, gastric carcinoma, multiple sclerosis, and
lymphomatoid granulomatosis. EBV not only evades
immune attack by infecting immune cells but has also
evolved other strategies to deal with it. EBV latent mem-
brane proteins are involved in activation and proliferation
of infected B cells. These proteins also reduce the activity
of CD8
?
T cells against EBV-infected cells [71]. Fur-
thermore, EBV inhibits the differentiation of DCs derived
from cord blood monocytes and induces apoptosis in a
caspase-dependent manner [72].
EBV expresses two transcription factors, Rta and Zta, in
the immediate early stage of the lytic cycle, which activate
early and late gene transcription and are required for
completion of the lytic cycle. To inhibit EBV propagation,
it is necessary to inhibit the expression of these two pro-
teins. Andrographolide has been tested against EBV in a
Burkitt’s lymphoma cell line using chemical stimulators of
the lytic cycle. Lin et al. tested both A. paniculata (25 lg
of ethanolic extract per ml) and andrographolide (5 lg/ml),
and both of them were found to inhibit EBV lytic proteins,
i.e., Rta, Zta and EA-D. Further studies revealed that the
lack of expression of these lytic proteins was due to inhi-
bition of transcription of two immediate-early genes:
BRLF1 (which codes for Rta) and BZLF1(which codes for
Zta) [73].
Andrographolide as an antiviral agent
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Human papillomavirus (HPV)
Human papillomavirus is a small non-enveloped DNA
virus with a diameter of about 55 nm. HPV belongs to the
family Papillomaviridae and genus Alphapapillomavirus.
The HPV capsid is icosahedral in symmetry, and contains a
double-stranded circular DNA genome of about 8 kb. The
HPV genome consists of three regions, namely, the early
region, encoding NS genes involved in virus replication
and inhibition of tumor suppressor proteins, the late region,
encoding structural genes, and the regulatory region, con-
taining the origin of DNA replication and elements regu-
lating DNA replication. HPV is transmitted through
vaginal, anal and oral sex and enters cells via clathrin-
mediated endocytosis. HPV infects basal epithelial cells
through wounds and abrasions and causes genital warts and
cancer in the cervical, vulvar, vaginal, penile, anal, and
oropharyngeal regions.
HPV in order to escape from immune attack, affects the
differentiation of immune cells, leading to immune toler-
ance. The modifications induced by HPV include tumor-
associated macrophage differentiation, a compromised
cellular immune response, Th1-Th2 cell imbalance, T
reg
infiltration, and downregulation activation and maturation
of DCs [74]. However, vaccines based on delivery of the
L1 gene of HPV (AcHERVHPV) have been screened in
cell lines and mouse models and found to elicit a strong
cellular and humoral immune response [75]. Further
compounds suppressing the viral genes and revitalizing the
tumor suppressor proteins can provide the promising
antiviral effects against HPV.
Andrographolide has been tested for anti-HPV activity.
Ekalasananan et al. examined the effects of andro-
grapholide and its derivatives IPAD and 14-DDA on
HPV16 pseudovirus (HPV16PsVs), HPV E6 oncogene
expression, and cervical cancer cell apoptosis [76]. They
reported that the inhibitory effects of andrographolide and
its derivatives on HPV16 infection include inhibition of the
E6 oncogene, restoration of p53 tumor suppressor protein,
and induction of cervical cell apoptosis. They also reported
that andrographolide and its derivatives prevented the
binding of HPV16PsVs to host-cell receptors, and 14-DDA
showed the highest potency of post-attachment inhibition.
Human immunodeficiency virus (HIV)
Human immunodeficiency virus belongs to the family
Retroviridae and genus Lentivirus. HIV is a positive-sense,
enveloped RNA virus containing two identical copies of a
single-stranded RNA genome, each of 9181 bp. HIV is a
spherical virus with a diameter of about 120 nm. In the
early stages, HIV infection is M- tropic, i.e., infecting
macrophages (including monocytes, DCs and microglial
cells) expressing the CD4 receptor and CCR5 coreceptor.
In the middle phase of infection, HIV is dual tropic, i.e.,
infecting macrophages and T cells expressing CD4, CCR5
and CXCR4. At the late phase of infection, HIV is T-tropic,
i.e., showing preference for T cells expressing the CD4 and
CXCR4 receptors. HIV is transmitted through unprotected
sex, blood transfusion, and infected needles. Mother-to-
child transfer of HIV can take place during pregnancy,
labour, delivery, and breast feeding. Infection of immune
cells with HIV leads to acquired immunodeficiency syn-
drome (AIDS). Along with pain, fever and other virus-
associated symptoms, HIV patients may develop oppor-
tunistic infections due to a weakened immune system [77].
Combinations of antiviral drugs are given to HIV
patients (anti-retroviral therapy), which reduce HIV-in-
duced morbidity and mortality. However, emergence of
viral resistance has shifted the focus towards developing
new drugs against HIV. Various studies have been carried
out to test the anti-HIV activity of andrographolide. Chang
et al. reported the anti-HIV activity of a succinyl derivative
of andrographolide, dehydroandrographolide succinic acid
monoester (DASM), in H9 cells and human PBMCs [78].
They found that DASM exhibits anti-HIV effects by
interfering with the binding of HIV virions to cells and by
obstructing a step in the viral replication cycle subsequent
to virus-cell binding. Calabrese et al. conducted a phase I
trial of andrographolide in HIV-infected patients and found
a significant increase in CD4
?
lymphocytes on treatment
with andrographolide. The higher dose of andrographolide
also reduced the HIV RNA copy number, but this decrease
was not significant [79]. This study showed that andro-
grapholide, rather than inhibiting the virus directly, inhibits
the virus-induced dysregulation of cell signaling pathways.
p24 antigen is a viral protein that makes the viral capsid
or core, and its expression is highest during the early phase
of infection p24 antigen. Reddy et al. reported that
andrographolide and other compounds isolated from A.
paniculata reduced the level of p24 antigen in MT2 cell
line [80].
Chikungunya virus (CHIKV)
Chikungunay virus belongs to the family Togaviridae,
genus Alphavirus. It is an enveloped single-stranded posi-
tive-sense RNA virus with a diameter of about 60-70 nm.
The RNA genome, which is about 11.805 kb long, is
capped at the 50end and has a 30polyA tail. The 49S
CHIKV genome is first translated to produce NS proteins,
which form the viral replicase and help in RNA genome
replication and formation of 26S subgenomic RNA for
structural protein translation [81].
Chikungunya virus is an arbovirus that is spread by
Aedes mosquitoes. When an infected mosquito bites its
S. Gupta et al.
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primate host, CHIKV enters the host through the mosquito
saliva and starts its replication in epithelial, endothelial,
fibroblast, monocyte and macrophage cells [82]. CHIKV
targets the joints, muscle, and skin, and rarely, the liver,
kidneys, eyes and central nervous system (CNS). A blood
meal from the infected host allows further transmission of
the virus to an uninfected mosquito, which can later
transmit CHIKV to a new host. CHIKV infection leads to
the fever, headache, rashes, myalgia, and polyarthralgia,
and the symptoms of myalgia and arthralgia can last for
years.
Due to the lack of effective anti-chikungunya drugs, only
the symptoms of disease can be treated. Wintachai et al.
reported the anti-CHIKV activity of andrographolide in
HepG2 and BHK-21 cells and found a decrease in the
CHIKV RNA copy number and CHIKV protein expression
[83]. They showed that andrographolide exerts an anti-
CHIKV effects at a post-entry step and effectively inhibits
viral genome replication. Our unpublished data also supports
the in vitro and in vivo anti-CHIKV activity of andro-
grapholide. We conducted our study on human monocytic
cells and in mouse neonates, and both experimental models
confirmed the potency of andrographolide as anti-CHIKV
agent. Moreover, we also observed immunomodulatory
activity of andrographolide in CHIKV-infected human
PBMCs and the results showed a strengthening effect of
andrographolide on host innate immune cells. Andro-
grapholide induced RIG-1 and PKR expression in CHIKV-
infected cells, highlighting its anti-CHIKV effects and
antiviral mechanism of action against CHIKV infection.
Future perspectives and conclusion
Global warming, climate change, drought, flood, host
migration, and vector distribution provide pressure for the
emergence of new viruses. Emerging and re-emerging viral
diseases have become a global threat. Therefore, there is an
urgent need to control viral infections, making it necessary
to understand the mechanisms that viruses use to evade the
host immune system and the manner in which they over-
take and utilize the host machinery to replicate and persist.
Antiviral vaccines stimulate immunity against viruses
by the administration of live attenuated virus or viral
subunits or peptides. However, these vaccines lose their
efficacy if the antigenicity of the virus changes. Moreover,
vaccines have limited therapeutic effects. Plant-based
Fig. 3 Inhibitory effects of andrographolide on the viral life cycle
Andrographolide as an antiviral agent
123
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antiviral drugs have provided great hope for combating the
viral infection (Fig. 3). They target viral receptor and co-
receptor binding [84–87], fusion and adsorption to the cell
[88–93], virus replication [94,95], reverse transcription
and integration [96–98], viral protein translation [99] and
post-translational modifications [100,101]. The prevalence
of infectious diseases in developing countries requires cost-
effective antiviral drugs with high efficacy. Some plant-
derived antiviral drugs fulfill this challenge by being cost-
effective, easily available, low in cytotoxicity, and thera-
peutically effective against viral infections. Andro-
grapholide, a plant-derived compound is widely
distributed, is cost-effective, has low cytotoxicity and has
been shown to have antiviral activity against a number of
viral infections. However, much research is needed to
identify its target molecules in the viral life cycle.
Recently, a whole extract of A. paniculata was tested
against dengue virus and simian retrovirus infections
[102,103]. Moreover, Edwin et al. also suggested a role for
andrographolide in the management of the dengue virus
and chikungunya virus vector Aedes aegypti.[104].
Andrographolide assists in vector management by reducing
oviposition, causing CPE in the midgut epithelium, and
increasing larvicidal activity. However, additional research
is required to establish the role of this bioactive compound
in other viral infections. Plants are a good source of
bioactive compounds for antiviral drug development
[105,106], and recent studies on the unfolded protein
response pathway suggest that it could be an important
target of various plant-derived drugs [107–111]. Andro-
grapholide, being a strong immunomodulator, can also be
tested in combination therapies to treat infectious diseases.
Andrographolide appears to be effective against a variety
of viral infections, and in the future, it can be used in drug
development, either alone or in combination, for the inhi-
bition of virus infection and treatment of infectious
diseases.
Acknowledgements This work was supported by the Council of
Scientific and Industrial Research (CSIR) in the form of a research
fellowship granted to SG and the Defence Research and Development
Organisation (DRDO) in the form of a project grant.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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