Received: 1 August 2020 Revised: 5 October 2020 Accepted: 7 October 2020

DOI: 10.1002/pca.3011

RESEARCH ARTICLE

Immunoprotective potential of Ayurvedic herb Kalmegh

(Andrographis paniculata) against respiratory viral infections –
LC–MS/MS and network pharmacology analysis

Subhadip Banerjee1 | Amit Kar1 | Pulok K. Mukherjee1,2 | Pallab K. Haldar1 |

2 3
Nanaocha Sharma | Chandra Kant Katiyar

1
School of Natural Product Studies,
Department of Pharmaceutical Technology,
Jadavpur University, Kolkata, India
2
Institute of Bioresources and Sustainable
Development, An Autonomous Institute under
Department of Biotechnology, Government of
India, Imphal, India
3
Health Care Division, Emami Limited 13,
Kolkata, India
Correspondence
Pulok K Mukherjee, PhD, FRSC, FNASC,
Institute of Bioresources and Sustainable
Development, An Autonomous Institute under
Department of Biotechnology, Government of
India, Imphal-795001, India.
Email: naturalproductm@gmail.com;
director.ibsd@nic.in
Funding information
Department of Biotechnology, Grant/Award
Number: BT/PR40374/TRM/120/484/2020;
Ministry of Science and Technology
Abstract
Introduction: Immunity boosting has emerged as a global strategy to fight the
SARS-CoV-2 pandemic situation. In India, AYUSH systems of medicine have been
promoted as an immune-protection strategy. Andrographis paniculata (Burm. F) Nees
(AP) mentioned in Ayurveda has been widely used for treating sore throat, flu, and
upper respiratory tract infections which may provide possible novel therapeutic
approaches, exclusively targeting SARS-CoV-2 and its pathways.
Objectives: The present work uses liquid chromatography–tandem mass spectrome-
try (LC–MS/MS) metabolomics and combination synergy analysis based on network
pharmacology to mine multimode evidence to understand the possible mechanism of
action, diseases association, protein–protein interaction and major pathways involved
therein.
Material and methods: Metabolite profiling was performed by Agilent QTOF
LC–MS/MS system. Network pharmacology analysis was performed by using
functional annotation analysis based on databases like Binding DB, STRING, DAVID
and KEGG for further data mining. Further combination synergy was evaluated using
“neighbourhood approach” and networks were constructed through Cytoscape 3.2.1.
Results: The molecules from kalmegh provides immune-protection and anti-viral
response via involving different pathways, like toll-like receptor pathway, PI3/AKT
pathway and MAP kinase pathways against COVID-19 infection. The KEGG analysis
showed that in a vast majority of the most enriched pathways, AP were associated
with viral infections and upper respiratory tract infections.
Conclusions: The results suggest a synergy between andrographolide and other
molecules identified as safe and efficacious anti-inflammatory agent having effects
on upper respiratory tract infections and can significantly decrease the production of
cytokines and pro-inflammatory factors in viral infections.

KEYWORDS

Andrographis paniculata, immunoprotection, LC–MS/MS, network pharmacology, SARS-CoV-2

Phytochemical Analysis. 2020;1–11. wileyonlinelibrary.com/journal/pca © 2020 John Wiley & Sons, Ltd. 1
2 BANERJEE ET AL.
1 | I N T RO DU CT I O N
The COVID-19 pandemic has become a global problem. Millions of
people, including many medical workers, have been infected, and
thousands have died. Due to an incubation period of 14 days or lon-
ger, patients can cause multiple infections before symptoms become
observable. An effective cure is yet to be developed. The Ministry of
AYUSH, Government of India has advised self-care guidelines for pre-
ventive health measures and boosting immunity with special refer-
ence to respiratory health. Report suggests that traditional Indian
medicinal plants may provide possible novel therapeutic approaches,
exclusively targeting SARS-CoV-2 and its pathways.1 Andrographis
paniculata (Burm. F) Nees (AP), generally known as “king of bitters,” is
an herbaceous plant in the family Acanthaceae. In China, India,
Thailand, and Malaysia, this plant has been widely used for treating
sore throat, flu, and upper respiratory tract infections. The earliest
Ayurvedic sages named the plant accordingly, calling it kiratatikta,
which means “the bitter herb of the Kirata people.” Kalmegh has
received additional names since, and they have followed suit:
bhunimba is Sanskrit for “neem of the earth,” referring to its bitter
neem-like taste and effects, and its common name is “king of the
bitters”.2 In China it is known as “Chuan-Xin-Lian”; in Thailand it is
known as “Fah Tha Lai”; in Malaysia it is known as “Hempedubumi”; in
Japan it is known as “Senshinren”; and in Scandinavian countries it is
known as “green chiretta”.3 Extracts of this plant and andrographolide
exhibit pharmacological activities such as those that are immuno-
stimulatory, antiviral, and antibacterial. AP is a well-known anti-
inflammatory and anti-bacterial herb and its major constituent
andrographolide exhibits a broad range of biological activities, such as
anti-inflammatory, antibacterial, antitumour, antidiabetic, antimalarial,
4,5
and hepatoprotective. In the literature, clinical trials suggests that
AP has a strong treating capacity of viral respiratory infections.6–8 It
was noted that AP suppressed increased NOD-like receptor protein
3 (NLRP3), caspase-1, and interleukin-1β molecules which are
extensively involved in the pathogenesis of SARS-COV and likely
SARS-CoV-2 as well.1 Andrographolides have shown different
anti-viral activities against H5N1 influenza infection,9 with immuno-
stimulant activity10 effective against upper respiratory tract infec-
11
tion. It is also found to be effective in influenza A virus-induced
inflammation in a murine model through NF-κB and JAK-STAT
signalling pathway.12
Metabolomics is an important tool for standardisation and quality
13
control in Ayurveda. The combinatory treatments with herbal
extracts used in Ayurveda may achieve more stable “network
responses” than single isolated compound due to synergy or
“samyoga”.14 Ethnopharmacology is an important way towards dis-
covery of novel drugs and formulation from the multiple bioactive
components within the herbs and herbal medicinal extract.15 Network
pharmacology presents a systems perspective to drug discovery.16,17
This model of combination synergy based on liquid chromatography–
tandem mass spectrometry (LC–MS/MS) and network analysis using
the neighbourhood approach has been reported in other pharmaco-
logical domains to understand the mechanism of action of herbal
extracts used in traditional medicine by our group.18,19 Our present
study is based on the evaluation of the chemical and biological basis
for the multiple mechanisms of actions, which ameliorate stress at dif-
ferent levels of immune systems with close correlation to severe
acute respiratory syndrome (SARS) infections like COVID-19.
LC–MS/MS quadrupole time-of-flight (QTOF) analysis of the root
extract was performed. The molecules from LC–MS/MS analysis were
used to understand the multimode cross-validated network pharma-
cology underlying pathways, genes, protein–protein interaction (PPI)
study to elucidate the multi-component synergistic mechanism based
on the neighbourhood approach. It will help to understand the
mechanistic targets of molecules in AP root with the key mediators of
the immunprotective response and understand the intracellular and
extracellular levels of network signalling with respect to management
of COVID-19.
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Instrumentation and reagents
LC–MS/MS was performed using an Agilent LC-QTOF 6500 system
(Agilent Technologies, Santa Clara, CA, USA). A Milli-Q water purifica-
tion system (Millipore, Bedford, MA, USA) equipped with a 0.22 mm
Millipak Express filter and an Eyela (Tokyo, Japan) rotary vacuum
evaporate was used. Membrane filters (0.45 μm pore size) (Millipore)
were used for filtration of the mobile phase and syringe filters
(NYL 0.45 μm) were used for the filtration of the sample solution.
Methanol (HPLC grade), formic acid (HPLC grade) were procured from
Merck (Mumbai, India). All the other solvents (AR grade) procured
from Merck.
2.2 | Plant collection and extraction
Dried leaves of AP were procured from vendors of Kolkata, West
Bengal, India. The voucher specimen of the dried plant root (specimen
number SNPS/JU/2018/1088) was deposited at the School of Natural
Product Studies, Jadavpur University, and Kolkata, India. The leaves
were thoroughly extracted using a percolation method using ethanol
then filtered. The liquid extract obtained was concentrated at less
than 700 mm Hg pressure in a rotary evaporator. A yield of 4% (w/w)
was obtained. The extracts were dissolved in methanol. The solutions
were filtered through a Millipore filter (0.45 μm).
2.3 | LC-ESI-MS/MS analyses
LC–MS/MS was performed using an Agilent LC-QTOF 6500 system
with an Agilent ZORBAX Eclipse XDB column – C18 (2.1 mm ×
50 mm, 1.8 μm). The mobile phase consisted of water (0.1% formic
acid), acetonitrile (with 0.1% formic acid) was used as eluents in a
gradient mode (see Table 1). The injection volume was 20.00 μL and
BANERJEE ET AL. 3

T A B L E 1 Results of liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) analysis of Andrographis
paniculata roots

Sl no. Name Formula Precursor Observed mass Diff (ppm) Score Retention time
1 Rosmarinic acid C18H16O8 383.0735 360.0842 −0.9 84.5 2.382
2 2,4-Dihydroxycinnamic acid C9H8O4 181.0497 180.0423 0.48 81.92 2.636
3 Andrographidine F C25H28O13 559.1426 536.1533 0.62 78.26 3.088
4 p-Coumaroyltyrosine C18H17NO5 345.1452 327.1116 2.91 80.53 3.106
5 Andrographiside C26H40O10 530.2958 512.2619 −0.57 76.93 3.621
6 Andrographidine B C23H24O12 493.1344 492.1276 1.62 85.93 3.801
7 Andrographidine A C23H26O10 485.1424 462.1532 1.4 78.08 4.078
8 Ninandrographolide C26H40O9 519.257 496.2681 1.7 73.99 4.174
9 14-Deoxyandrographolide C20H30O4 335.222 334.2148 1.06 99.56 4.182
10 Andrograpanin C20H30O3 319.2272 318.2199 1.3 84.33 4.592
11 5-Hydroxy- 7,8,20 -trimethoxyflavone 5- glucoside C24H26O11 491.1557 490.1498 4.73 97.45 5.088
12 Andrographin C18H16O6 329.1019 328.0947 0.19 98.02 5.884
0 0
13 5-Hydroxy-7,8,2 ,3 -tetramethoxyflavone C19H18O7 359.1129 358.1055 0.55 98.62 6.011
14 Andrographidine D C23H24O10 483.1276 460.1382 2.74 73.1 6.094
15 Piceatannol C14H12O4 262.1074 244.0734 −0.78 81.79 6.209
16 Apigenin C15H10O5 271.0606 270.0533 1.77 99.06 6.675
17 Andrographolide C20H30O5 350.2329 332.1989 0.39 82.28 8.842
18 4-hydroxyphenylacetic acid C8H8O3 153.0549 152.0476 1.66 75.01 10.669
19 Methoxyphenylacetic acid C9H10O3 167.0704 166.0631 0.56 83.61 10.671
20 Pterostilbene C16H16O3 257.1175 256.1102 1.12 76 11.767
21 Resveratrol C14H12O3 229.0865 228.0792 2.64 80.75 14.042
22 Gardenin A C21H22O9 419.1341 418.1267 0.87 99.41 14.511

the column temperature was maintained at 30
C. The flow rate was
kept constant throughout the gradient. The ultra-performance liquid
chromatography (UPLC) system was coupled to a QTOF mass spec-
trometer (6500 series; Model-G6545B) equipped with an AP-ESI
Dual Spray source. Parameters for analysis were set using positive
ion mode with spectra acquired over a mass range from m/z 120 to
1000. The MS data were processed through MassHunter v B.08.00,
Rapid Control version 2.9 software (Agilent Technologies), which
provided a list of possible elemental formulae by integrating with
libraries. The accuracy for confirmation of the compounds was
established on the basis of an error less than 5 ppm and MS/MS
fragment matching.
2.4 | Network pharmacology analysis
2.4.1 | Target identification of bioactive
phytoconstituents and oral bioavailability screening
The target of each phytoconstituents was retrieved using a similar-
ity ensemble approach (SEA) from http://sea.bkslab.org/ which
relates proteins based on the set-wise chemical similarity among
20
their ligands. The gene code of each target was retrieved from
the UniProt database (https://www.uniprot.org/). All the targets of
each phytoconstituent involved were identified with reference to
the human targets for their accurate UniProtKB/ID (http://www.
uniprot.org/).
2.4.2 | Construction of target PPI, gene diseases
association network, and pathway analysis
PPI of the protein targets were searched from STRING 3.021 and
interactive visualisation with statistical analysis was done in
NetworkAnalyst (http://www.networkanalyst.ca).22 The associated
proteins and connected genes with the associated human disease
conditions were analysed from DisGeNET (http://www.disgenet.org)
to understand its potential link with COVID-19.23 The significant
nodes or targets were mapped to corresponding diseases from the
interaction database based on their betweenness centrality. Further
module analysis was performed to understand the most significant
association. The functional annotation of the molecule targets and
their role in signal transduction was performed with the Database for
Annotation, Visualisation and Integrated Discovery (DAVID 6.8).
2.4.3 | Combination synergy network analysis
Combination synergy of the molecules related to adaptogenic poten-
tial were analysed considering the “immunomodulatory” domain,
4 BANERJEE ET AL.
which were identified based on neighbourhood topology24 detailed in
our previous report.18 The networks were constructed in Cytoscape25
(http://cytoscape.org/, version 3.4.0) and web-based platform like
NetworkAnalyst.22
3 | RESULTS
3.1 | Metabolite profiling of AP leaf constituents
Figure 1 illustrates the total ion chromatogram (TIC) of liquid chroma-
tography triple quadruple mass spectrometry (LC-QqQ-MS) study.
We processed the chromatogram through MassHunter B.08 software,
which provided a list of 20 compounds (shown in Table 1). This ide-
ntified compounds with mass spectrometric data on experimental and
calculated m/z, error in parts per million (ppm), molecular formula,
retention time and MS fragmentation pattern (matched within error
tolerance of 25 ppm) with the proposed name of the compounds. The
TOF-MS method presents highly accurate observations with a mass
error of below 5 ppm. Terpenoid lactones and flavonoids have been
identified in the LC–MS/MS analysis as reported previously.26
Analysis of the ethanolic extract identified terpenoid lactones
like ninandrographolide, 14-deoxyandrographolide, andrograpanin,
andrographin, andrographolide were identified. Whereas, new several
related flavones glycosides andrographidine F, andrographiside,
andrographidine B, andrographidine A, 5-hydroxy- 7,8,20 -
trimethoxyflavone 5-glucoside, andrographidine D were identified.
Flavonoids like rosmarinic acid, piceatannol, apigenin, pterostilbene,
resveratrol, gardenin A have been found in the study which are crucial
to pharmacological activity.
3.2 | Target screening of molecules from AP leaves
The identified targets were searched using an SEA which relates pro-
teins based on the set-wise chemical similarity among their ligands
with respect to the AP compounds found from the metabolomics
study which were used as queries.19 The interacting targets and com-
pounds have been used for construction of the target-compound net-
work shown in Figure 2 with a network density of 0.01 (200 nodes;
290 edges); characteristics path length 1.0 and 2.9 average number of
neighbours. In this network, we found that many targets interact with
multiple compounds and vice-versa. Among the 17 compounds
we found apigenin, resveratrol and its metabolite piceatannol to
be interacting with most of the targets followed by pterostilbene
and rosmarinic acid, their closeness centrality is around 0.3.
Andrographidine D, B, F, and A and andrographin were also found to
be very crucial molecules interacting with interleukin-2 (IL-2) and simi-
lar targets which are connected to immunomodulation due to its
neighbourhood connectivity of 9.6. We found that andrographolide
majorly interacts with protein kinases which are implicated in various
other pathways representing the host–virus interactome. We found
p-coumaroyltyrosine to be an interaction angiotensin-converting
enzyme (ACE) which is a major target in COVID-19.26
3.3 | Target disease association network of
compounds from AP
Network-based target–disease proximity sheds light on the relation-
ship between drugs (e.g. drug targets) and disease modules (molecular
determinants in disease pathobiology modules within the PPIs). This
disease interaction model was created based on the targets screened
from the compounds and their high degree of association and
betweenness parameters in the network. It can be observed that this
disease association of the targets show high correlation that combines
synergy mechanism of the molecules together to contribute to the
immunomodulatory potential of AP in “immunomodulatory” domain.
The gene-disease association network (Figure 3) shows targets like
FABP5, FGF1, GLI1, IL2, MAP 2 K3, PIK3CB, ROS1, TLR9, TLR4,
CCR4 which are associated with disease terms like viral infections,
immunodeficiency, influenza, respiratory tract infections, are closely
F I G U R E 1 Total ion
chromatogram of LC-ESI-MS/MS
of Andrographis paniculata extract
BANERJEE ET AL.
F I G U R E 2 Compound target network
of compounds from Andrographis
paniculata. The targets are shown as blue
rectangles and the compounds are shown
as red diamonds
related with SARS in the disease gene association database which has
close similarity with COVID-19.27
3.4 | Construction and analysis of target PPI
network
The PPI study was used to identify proteins that associate with and
functionally govern viral infection localised in the signalling pathways,
and networks which represent the human corona virus (HCOV)–host
interactome to understand the mechanism of the compounds from
AP. Molecular functions and pathways of target proteins were
analysed based on PPI experimental data from the STRING database
interactome.21 A P-value of < 0.05 was considered for building a
5
diversified network. We obtained the enrichment analysis functional
gene ontologies (GOs) and pathway enrichment analysis from KEGG.
The PPI network showed that protein targets like STAT3, JUN,
PRCKQ, PRCKB, and PRCKH have higher interactions between them.
The GO-process analysis confirmed the role of these proteins in
transferase activity, transferring phosphorus-containing groups
(GO:0016772), nucleotide binding (GO:0000166), adenyl ribonucleo-
tide binding (GO:0032559), purine ribonucleotide binding
(GO:0032555), etc. The Reactome pathway enrichment was searched
which retrieved some significant pathways shown in Table 2. Differ-
ent pathways like Signalling by Interleukins (HSA-449147), Nuclear
Receptor transcription pathway (HSA-383280), Cytokine Signalling in
Immune system (HSA-1280215), Interleukin-4 and Interleukin-13
signalling (HSA-6785807) showed significant enrichment which may
6 BANERJEE ET AL.
FIGURE 3 Disease target association network (the targets are shown as blue rectangles and the compounds are shown as purple circles)
be important in HCOV–host interaction protein targets based on PPI.
The KEGG enrichment showed its potential role in TH17 cell
differentiation (hsa04659).
The proteins were further visualised to identify the nodes that
represent the module or subnetwork which are important in order to
understand more about the mechanism of action of the AP molecules
as shown in Figure 4. The larger nodes are the hub proteins connected
to host protein targets which may act as COVID-19 targets.28 We
analysed the query protein sets of the PPI analysis for KEGG path-
ways. Analysing the KEGG database based on the PPI pathways with
low P-value and false discovery rate are shown in Table 3. Several evi-
dences have been identified at the pathway level which strongly
suggests that an AP molecule combats stress at cellular, tissue and
organ system level. It was found that the pathways implicated in
immune-protection potential are all implicated in immune response
and may be effective in COVID-19 infections.29 These pathways have
also been highlighted by the traditional Chinese formulation found to
be effective against COVID-19.30
3.5 | DAVID pathway analysis
The molecular targets of WS were further analysed for functional
association clustering to integrate functional genomics annotations of
the most important cluster of targets, pathways, domains highlighting
the immunomodulatory action of the AP target proteins of host
induced by HCOV–host interaction. The results showed that the
molecule in AP interacts with the toll-like receptor signalling pathway
as shown in Figure 5. The marked stars (see Figure 5) signify the
protein targets involved in the pathways like TLR9, TLR4, PI3K, JNK,
IL-8, etc. The compound target network and pathway showed,
andrographolide, andrographodin A interacts with important targets
known as TLR4, TLR, IL-8, etc. The earlier-mentioned PPI analysis
showed high enrichment of PI3k-AKT pathway. The red stars in
Figure 6 show that the AP molecules interact with targets like PI3k,
TLR4, CREB, etc. Evidence shows PI3K-AKT signalling responses play
important roles in Middle East respiratory syndrome-related
coronavirus (MERS-CoV) infection which is closely associated with
COVID-19 and may represent novel drug targets for therapeutic
intervention strategies.31
3.6 | Combination synergy by network analysis
The combination synergy mechanism of action of the molecular com-
binations of AP to evaluate its potential to combat respiratory tract
infection of viral origin has been analysed using a deductive approach
where the interacting targets and the associated disease conditions
have been studied using network pharmacology methods. We devised
a simple neighbourhood network connection analysis as described in
the Methods section. We show the combination synergy networks
related to the disease domains (see Figure 7). In the individual combi-
natory mechanism deconstructing that network showed the molecules
interacting with the targets associated to the respective pharmacolog-
ical activity were connected to the viral infections and respiratory
tract infections. We found that 16 compounds together interacted
with 28 targets connected with COVID-19 associated conditions like
immunodeficiency, asthma, influenza, viral infections, respiratory
BANERJEE ET AL. 7

TABLE 2 Reactome pathway enrichment analysis based on protein–protein interactions (PPIs)

False
#term ID Term description discovery rate Matching proteins in your network (labels)
HSA-383280 Nuclear receptor transcription pathway 2.25E-11 AR, ESR1, ESR2, ESRRA, ESRRB, ESRRG, HNF4A,
NR0B2, PPARA, PPARD, PPARG, THRA, THRB,
VDR
HSA-1280215 Cytokine signalling in immune system 2.94E-11 ALOX15, ALOX5, APP, CA1, CAMK2A, CASP1,
CDKN1A, CREB1, CXCL8, FGF2, FLT3, FYN,
HCK, IKBKG, IL2, JAK2, JAK3, LGALS9, MAOA,
MAP 2 K3, MAPK10, MCL1, MIF, MMP1, MMP2,
MMP9, NFKB1, P4HB, PIK3CA, PIK3CB, PIM1,
PPM1B, PRKACA, PTAFR, PTGS2, PTPN6, RELA,
RPS6KA2, RPS6KA3, SYK, TBK1, TNFRSF1A,
VEGFA
HSA-449147 Signalling by interleukins 7.82E-15 ALOX15, ALOX5, APP, CA1, CASP1, CDKN1A,
CREB1, CXCL8, FGF2, FLT3, FYN, HCK, IKBKG,
IL2, JAK2, JAK3, LGALS9, MAOA, MAP 2 K3,
MAPK10, MCL1, MIF, MMP1, MMP2, MMP9,
NFKB1, P4HB, PIK3CA, PIK3CB, PIM1, PRKACA,
PTAFR, PTGS2, PTPN6, RELA, RPS6KA2,
RPS6KA3, SYK, TBK1, TNFRSF1A, VEGFA
HSA-168249 Innate immune system 1.30E-08 ABL1, ACPP, ALOX5, ANPEP, APP, ASAH1,
B4GALT1, BTK, CASP1, CDK13, CPN1, CREB1,
CTSV, DNM1, EP300, FABP5, FYN, HCK, HRAS,
IKBKE, IKBKG, LGALS3, LTA4H, MAP 2 K3,
MAPK10, MIF, MME, MMP9, NFKB1, NRAS,
PIK3CA, PIK3CB, PRKACA, PRKCQ, PTAFR,
PTK2, PTPN6, RELA, RHOA, ROCK1, RPS6KA2,
RPS6KA3, SELL, SLCO4C1, SYK, TBK1, TLR4,
TRPM2, TTR
HSA-9018678 Biosynthesis of specialised proresolving mediators 2.54E-08 ALOX12, ALOX15, ALOX5, CYP1A1, CYP1A2,
(SPMs) CYP2C9, CYP3A4, LTA4H, PTGS2
HSA-6785807 Interleukin-4 and Interleukin-13 signaling 1.18E-07 ALOX15, ALOX5, CDKN1A, CXCL8, FGF2, JAK2,
JAK3, MAOA, MCL1, MMP1, MMP2, MMP9,
PIM1, PTGS2, VEGFA
HSA-166016 Toll-like receptor 4 (TLR4) cascade 4.16E-06 APP, BTK, CREB1, DNM1, IKBKE, IKBKG, MAP
2 K3, MAPK10, NFKB1, RELA, RPS6KA2,
RPS6KA3, TBK1, TLR4
HSA-168898 Toll-like receptor cascades 5.31E-06 APP, BTK, CREB1, CTSV, DNM1, IKBKE, IKBKG,
MAP 2 K3, MAPK10, NFKB1, RELA, RPS6KA2,
RPS6KA3, TBK1, TLR4
HSA-166166 MyD88-independent TLR4 cascade 9.15E-06 APP, CREB1, IKBKE, IKBKG, MAP 2 K3, MAPK10,
NFKB1, RELA, RPS6KA2, RPS6KA3, TBK1, TLR4
HSA-937061 TRIF (TICAM1)-mediated TLR4 signaling 9.15E-06 APP, CREB1, IKBKE, IKBKG, MAP 2 K3, MAPK10,
NFKB1, RELA, RPS6KA2, RPS6KA3, TBK1, TLR4
HSA-168164 Toll-like receptor 3 (TLR3) cascade 8.49E-06 APP, CREB1, IKBKE, IKBKG, MAP 2 K3, MAPK10,
NFKB1, RELA, RPS6KA2, RPS6KA3, TBK1, TLR4
HSA-168188 Toll-like receptor TLR6:TLR2 cascade 4.42E-05 APP, BTK, CREB1, IKBKG, MAP 2 K3, MAPK10,
NFKB1, RELA, RPS6KA2, RPS6KA3, TLR4
HSA-451927 Interleukin-2 family signalling 5.24E-05 IL2, JAK2, JAK3, LGALS9, PIK3CA, PIK3CB, PTPN6,
SYK
HSA-166520 Signalling by NTRKs 5.47E-05 CDK5, CDK5R1, CREB1, DNM1, HRAS, NRAS,
PIK3CA, PIK3CB, RHOA, RPS6KA2, RPS6KA3
HSA-168179 Toll-like receptor TLR1:TLR2 cascade 5.47E-05 APP, BTK, CREB1, IKBKG, MAP 2 K3, MAPK10,
NFKB1, RELA, RPS6KA2, RPS6KA3, TLR4
HSA-181438 Toll-like receptor 2 (TLR2) cascade 5.47E-05 APP, BTK, CREB1, IKBKG, MAP 2 K3, MAPK10,
NFKB1, RELA, RPS6KA2, RPS6KA3, TLR4
8 BANERJEE ET AL.

F I G U R E 4 Protein–protein interaction
network of Andrographis paniculata

TABLE 3 KEGG pathway enrichment analysis based on protein–protein interactions (PPIs)

Pathway Total Expected Hits P Value False discovery rate

Toll-like receptor signalling pathway 97 0.85 7 1.67E-05 0.00362
Chemokine signalling pathway 189 1.66 8 0.000185 0.00803
B cell receptor signalling pathway 75 0.657 5 0.000442 0.0137
T cell receptor signalling pathway 98 0.859 5 0.0015 0.025
MAPK signalling pathway 265 2.32 8 0.00177 0.0256
Epstein–Barr virus infection 91 0.797 4 0.00782 0.0543
Measles 102 0.894 4 0.0116 0.0663
Influenza A 107 0.937 4 0.0137 0.0723

distress, etc. We found molecules like andrographolide,
andrographidine, andrographin, resveratrol, pterostilbene are essen-
tially responsible for these HCOV–host conditions, which are working
together on the targets MAPk3, RHOA, IL-2, MAPK10, ESR2, CREB2,
MMP12, etc. to combat the targets which are connected to
COVID-19-related genes as can be inferred from the earlier multiple
evidences of pathway analysis and PPI analysis.
4 | DISCUSSIONS
Diving deep into traditional knowledge of natural products based on
their use and mechanism can be an effective strategy of repurposing
existing resources in global pandemic conditions like COVID-19 which
can lead to traditional knowledge inspired discovery.15,29 AP is
deemed to be safe and used as medicinal food in Ayurveda by Indian
population with history of safe use.32 Metabolomics is an effective
way to decipher the intricate chemistry of natural products which is
the key to understanding the chemical basis of biological activity.13
Our study of ethanolic extract of AP on metabolite profiling by
LC–MS/MS analysis showed the presence of 22 compounds which
had strong correlation with similar reports previously.4 AP is known
for its activity in upper respiratory traction infection.11 Our study
shows the presence of 14-deoxy-11,12-dehydroandrographolide
which can effectively inhibit H5N1 replication by restraining nuclear
export of viral ribonucleoprotein complexes while minimising the
upregulated expression of pro-inflammatory cytokines/chemokines
induced by H5N1.9 It is also known to inhibit the syncytial virus that
causes respiratory tract infection titer similar to Lamivudine.10 The
major constituent andrographolide is known to inhibit influenza A
virus-induced inflammation in a murine model through NF-κB and
JAK-STAT signalling pathway.12 Clinical evidence shows AP shortened
the duration of cough, sore throat and sick leave/time to resolution
which suggests it has ameliorative potential and is safe in relieving
acute respiratory tract infection symptoms and shortening time to
symptom resolution.6 Using network pharmacological analysis, we
BANERJEE ET AL. 9

F I G U R E 5 Pathway mapping of Andrographis paniculata targets involved in toll-like receptor signalling pathway (the red stars signify the
molecules interacting at different targets of the signalling)

F I G U R E 6 Pathway mapping of Andrographis paniculata targets involved in PI3K-AKT signalling pathway.(the red stars signify the molecules
interacting at different targets of the signalling)
10
F I G U R E 7 Combination synergy network of Andrographis paniculata showing immunoprotection against viral and upper respiratory infection
(the targets are shown as blue rectangles and the compounds are shown as purple circles and red diamonds)
found that these components can cure or relieve the symptoms of
stress, rheumatoid arthritis, inflammation, etc. through the action of
target proteins in various pathways connected to immunomodulation.
Network pharmacology to understand combination synergy analysis
was also performed as a research strategy which provides a unique
and innovative path for understanding the mechanism of action of
herbs and its multi-components.18,19 The results indicated that AP can
suppress viral replication by inhibition of AKT, reduce apoptosis, and
thereby repair damage to the body as it perturbs the PI3K-Akt signal-
ling pathway.33 In SARS infected individuals the phosphorylation level
of Akt is downregulated in cells expressing M protein and
3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibits M
protein-induced apoptosis thereby inhibiting PDK1 and PKB/Akt cell
survival signalling pathway.34
The KEGG analysis showed that in a vast majority of the most
enriched pathways, AP was associated with viral infections and upper
respiratory tract infections. These pathways included the toll-like
influenza, Chemokine signalling, MAP-Kinase pathway, Epstein–Barr
virus infection, HTLV-I infection, and P13K/Akt signalling pathways
which have been identified as important targets for HCOV–host inter-
actions.35 Notably, the P13K/Akt pathway which meets the needs of
replication for many viruses and as the “proviral” kinase is activated, it
activates the host's response to viral infection and ultimately inhibits
36
viral replication. The study further suggests the importance of sev-
eral other Ayurvedic herbs mentioned in Ayurveda as Rasayana with
proven immunomdulatory effects should be explored for their poten-
tial in COVID-19 management.
The metabolite profiling and combination synergy analysis is a
prerequisite for the chemical and pharmacological investigations of
traditional medicines, as well as for their clinical applications. The
combination synergy analysis based on network pharmacology
showed multimode evidence that molecules from kalmegh provides
immune-protection based on the distinct pharmacological domain
largely. It works on the cell signalling pathway involving protein
kinase-c subtypes. From the pathway analysis, it was observed that
the different pathways like interleukins, Cytokine signalling which
connects the molecules to its immune-protective properties and anti-
viral response via modulation PI3/AKT and MAP kinase pathways
BANERJEE ET AL.
against COVID-19 infection. We suggest a synergy between
andrographolide and its chemo-similars identified as safe and success-
ful anti-inflammatory agents having effects on upper respiratory tract
infections and can significantly decrease the production of cytokines
and pro-inflammatory factors in COVID-19. Further our results
showed the necessity to perform suitable system biological experi-
ments and clinical trials to confirm the mechanism of WS against
COVID-19. The network analysis based on neighbourhood approach
can help in further understand the potential of AP and its components
in the management of SARS including COVID-19.
AC KNOWLEDG EME NT
The authors are thankful to the Institute of Bioresources and
Sustainable Development, an autonomous institute under Department
of Biotechnology (DBT), Government of India, Imphal, India for
necessary help and support through IBSD-JU joint collaboration. The
authors are also thankful to the Department of Biotechnology (Grant
no. BT/PR40374/TRM/120/484/2020) Ministry of Science and Tech-
nology, Government of India, New Delhi.
OR CID
Pulok K. Mukherjee https://orcid.org/0000-0002-2859-3923
RE FE RE NCE S
1. Vellingiri B, Jayaramayya K, Iyer M, et al. COVID-19: A promising cure
for the global panic. Sci Total Environ. 2020;725:138277. https://doi.
org/10.1016/j.scitotenv.2020.138277
2. Pole S. Ayurvedic Medicine: The Principles of Traditional Practice.
London, UK & Philadelphia, USA: Singing Dragon; 2012.
3. Ajaya Kumar R, Sridevi K, Vijaya Kumar N, Nanduri S, Rajagopal S.
Anticancer and immunostimulatory compounds from Andrographis
paniculata. J Ethnopharmacol. 2004;92(2):291-295.
4. Dai Y, Chen S-R, Chai L, Zhao J, Wang Y, Wang Y. Overview of phar-
macological activities of Andrographis paniculata and its major com-
pound andrographolide. Crit Rev Food Sci Nutr. 2019;59(sup1):
S17-S29.
5. Jayakumar T, Hsieh C-Y, Lee J-J, Sheu J-R. Experimental and Clinical
Pharmacology of Andrographis paniculata and Its Major Bioactive
Phytoconstituent Andrographolide. Evid Based Complement Alternat
Med. 2013;2013:1-16. https://doi.org/10.1155/2013/846740
BANERJEE ET AL.
6. Hu X-Y, Wu R-H, Logue M, et al. Andrographis paniculata (Chuan Xīn
Lián) for symptomatic relief of acute respiratory tract infections in adults
and children: A systematic review and meta-analysis. PLOS ONE. 2017;
12(8):e0181780. https://doi.org/10.1371/journal.pone.0181780
7. Chang J, Zhang R, Zhang Y, et al. Andrographolide drop-pill in treat-
ment of acute upper respiratory tract infection with external wind-
heat syndrome: A multicenter and randomized controlled trial. Zhong
Xi Yi Jie he Xue Bao. 2008;6(12):1238-1245.
8. Poolsup N, Suthisisang C, Prathanturarug S, Asawamekin A,
Chanchareon U. Andrographis paniculata in the symptomatic treat-
ment of uncomplicated upper respiratory tract infection: Systematic
review of randomized controlled trials. J Clin Pharm Ther. 2004;29(1):
37-45.
9. Cai W, Li Y, Chen S, et al. 14-Deoxy-11,12-dehydroandrographolide
exerts anti-influenza A virus activity and inhibits replication of H5N1
virus by restraining nuclear export of viral ribonucleoprotein com-
plexes. Antiviral Res. 2015;118:82-92.
10. Churiyah, Pongtuluran OB, Rofaani E, Tarwadi. Antiviral and immuno-
stimulant activities of Andrographis paniculata. HAYATI J Biosci. 2015;
22(2):67-72.
11. Panossian A, Wikman G. Efficacy of Andrographis paniculata in upper
respiratory tract infectious diseases and the mechanism of action. In:
Wagner H, Ulrich-Merzenich G, eds. Evidence and Rational Based
Research on Chinese Drugs. Vienna: Springer; 2013:137-179.
12. Ding Y, Chen L, Wu W, Yang J, Yang Z, Liu S. Andrographolide
inhibits influenza a virus-induced inflammation in a murine model
through NF-κB and JAK-STAT signaling pathway. Microbes Infect.
2017;19(12):605-615.
13. Mukherjee PK, Harwansh RK, Bahadur S, et al. Metabolomics of
medicinal plants – a versatile tool for standardization of herbal prod-
ucts and quality evaluation of Ayurvedic formulations. Curr Sci. 2016;
111(10):1624.
14. Mukherjee PK, Banerjee S, Kar A. Exploring synergy in Ayurveda and
traditional Indian systems of medicine. Synergy. 2018;7:30-33.
15. Mukherjee PK. Chapter 2 - Ethnopharmacology and ethnomedicine-
inspired drug development. In: Mukherjee PK, ed. Quality Control and
Evaluation of Herbal Drugs. Amsterdam, Netherlands: Elsevier; 2019:
29-51.
16. Hopkins AL. Network pharmacology. Nat Biotechnol. 2007;25(10):
1110-1111.
17. Hopkins AL. Network pharmacology: The next paradigm in drug dis-
covery. Nat Chem Biol. 2008;4(11):682-690.
18. Banerjee S, Bhattacharjee P, Kar A, Mukherjee PK. LC–MS/MS analy-
sis and network pharmacology of Trigonella foenum-graecum – A plant
from Ayurveda against hyperlipidemia and hyperglycemia with combi-
nation synergy. Phytomedicine. 2019;60:152944. https://doi.org/10.
1016/j.phymed.2019.152944
19. Goswami D, Mahapatra AD, Banerjee S, et al. Boswellia serrata oleo-
gum-resin and β-boswellic acid inhibits HSV-1 infection in vitro
through modulation of NF-кB and p38 MAP kinase signaling.
Phytomedicine Int J Phytother Phytopharm. 2018;51:94-103.
20. Keiser MJ, Roth BL, Armbruster BN, Ernsberger P, Irwin JJ,
Shoichet BK. Relating protein pharmacology by ligand chemistry. Nat
Biotechnol. 2007;25(2):197-206.
21. Szklarczyk D, Morris JH, Cook H, et al. The STRING database in
2017: quality-controlled protein–protein association networks,
made broadly accessible. Nucleic Acids Res. 2017;45(Database
issue):D362-D368.
22. Xia J, Gill EE, Hancock REW. NetworkAnalyst for statistical, visual
and network-based meta-analysis of gene expression data. Nat
Protoc. 2015;10(6):823-844.
11
23. Piñero J, Bravo À, Queralt-Rosinach N, et al. DisGeNET: A
comprehensive platform integrating information on human disease-
associated genes and variants. Nucleic Acids Res. 2017;45(D1):
D833-D839.
24. Zou J, Ji P, Zhao Y-L, et al. Neighbor communities in drug combina-
tion networks characterize synergistic effect. Mol Biosyst. 2012;8(12):
3185-3196.
25. Shannon P, Markiel A, Ozier O, et al. Cytoscape: A software environ-
ment for integrated models of biomolecular interaction networks.
Genome Res. 2003;13(11):2498-2504.
26. Kumar S, Singh A, Bajpai V, Sharma KR, Kumar B. Identification and
characterization of terpenoid lactones and flavonoids from ethanolic
extract of Andrographis paniculata (Burm.F.) Nees using liquid chro-
matography/tandem mass spectrometry. Sep Sci PLUS. 2018;1(12):
762-770.
27. Ledford H. How does COVID-19 kill? Uncertainty is hampering doc-
tors' ability to choose treatments. Nature. 2020;580(7803):311-312.
28. Gordon DE, Jang GM, Bouhaddou M, et al. A SARS-CoV-2 protein
interaction map reveals targets for drug repurposing. Nature. 2020;
583(7816):459-468.
29. Zhou Y, Hou Y, Shen J, Huang Y, Martin W, Cheng F. Network-based
drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2. Cell
Discov. 2020;6(1):1-18.
30. Yu S, Wang J, Shen H. Network pharmacology-based analysis of the
role of traditional Chinese herbal medicines in the treatment of
COVID-19. Ann Palliat Med. 2020;9(2):437-446-446-446.
31. Kindrachuk J, Ork B, Hart BJ, et al. Antiviral potential of ERK/-
MAPK and PI3K/AKT/mTOR signaling modulation for Middle East
respiratory syndrome coronavirus infection as identified by tempo-
ral Kinome analysis. Antimicrob Agents Chemother. 2015;59(2):
1088-1099.
32. Kar A, Pandit S, Mukherjee K, Bahadur S, Mukherjee PK. Safety
assessment of selected medicinal food plants used in Ayurveda
through CYP450 enzyme inhibition study. J Sci Food Agric. 2017;97
(1):333-340.
33. Mizutani T, Fukushi S, Saijo M, Kurane I, Morikawa S. Importance of
Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6
cells. Virology. 2004;327(2):169-174.
34. Tsoi H, Li L, Chen ZS, Lau K-F, Tsui SKW, Chan HYE. The
SARS-coronavirus membrane protein induces apoptosis via interfer-
ing with PDK1-PKB/Akt signalling. Biochem J. 2014;464(3):439-447.
35. Li X, Yu J, Zhang Z, et al. Network bioinformatics analysis
provides insight into drug repurposing for COVID-19. Preprints
2020, 2020030286. https://doi.org/10.20944/preprints202003.
0286.v1
36. Dunn EF, Connor JH. HijAkt: the PI3K/Akt pathway in virus replica-
tion and pathogenesis. Prog Mol Biol Transl Sci. 2012;106:223-250.
How to cite this article: Banerjee S, Kar A, Mukherjee PK,
Haldar PK, Sharma N, Katiyar CK. Immunoprotective potential
of Ayurvedic herb Kalmegh (Andrographis paniculata) against
respiratory viral infections – LC–MS/MS and network
pharmacology analysis. Phytochemical Analysis. 2020;1–11.
https://doi.org/10.1002/pca.3011