Network pharmacology of AYUSH recommended

immune-boosting medicinal plants against COVID-

19
Pukar Khanal 
Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi, KLE Academy of
Higher Education and Research (KAHER), Belagavi-590010, Karnataka, India
Taaza Duyu 
Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi, KLE Academy of
Higher Education and Research (KAHER), Belagavi-590010, Karnataka, India
Yadu Nandan Dey  (  [email protected] )
School of Pharmaceutical Technology, Adamas University, Kolkata-700126, West Bengal, India
B M Patil 
Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi, KLE Academy of
Higher Education and Research (KAHER), Belagavi-590010, Karnataka, India
Iasmail Pasha 
Department of Pharmacology, Orotta College of Medicine and Health Sciences, Asmara University,
Asmara, Eritrea
Manish Wanjari 
Regional Ayurveda Research Institute for Drug Development, Gwalior, Madhya Pradesh, India

Research Article

Keywords: AYUSH, COVID19, herbal tea, immunity promotion

DOI: https://doi.org/10.21203/rs.3.rs-31776/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  
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Abstract
Background: The Ministry of AYUSH recommended the use of a decoction of the mixture of Ocimum
tenui orum, Cinnamomum verum, Piper nigrum, Zingiber o cinale, and Vitis vinifera as a preventive
measure to boost immunity and to inhibit the severity of infection caused by a novel coronavirus (COVID-
19).

Objective: The present study aimed to identify the probable modulated pathways via AYUSH
recommended formulation as an immune booster against COVID-19.

Materials and methods: Reported phytoconstituents of all the plants were retrieved from the ChEBI
database, and their targets were predicted using DIGEP-Pred. STRING database and Cytoscape were used
to predict the protein-protein interaction and construct the network interaction respectively. Likewise,
MolSoft and admetSAR2.0 were used to predict the druglikeness score and ADMET pro le of
phytoconstituents.

Results: The study identi ed the modulation of HIF-1, p53, PI3K-Akt, MAPK, cAMP, Ras, Wnt, NF-kappa B,
IL-17, TNF, and cGMP-PKG signaling pathways to boost the immune system. Further, multiple pathways
were also identi ed which are involved in the regulation of pathogenesis of the multiple infections and
non-infectious diseases due to the lower immune system.

Conclusion: Results indicated that the recommended herbal formulation not only modulated the
pathways related to boost the immune system but also modulated the multiple pathways that are
contributing in the progression of multiple disease pathogenesis which would add the bene cial effect in
the special subjects like patients from hypertension and diabetes in which 4-hydroxychloroquine
therapeutic approach cannot be made. The study provides the scienti c documentation of the role of the
Ayurvedic formulation to combat COVID-19. 

1. Introduction
In the last 6 months, the novel coronavirus (COVID-19) has spread over the globe infecting more than 2
million populations leading to more than 100 thousand deaths. Subjects suffering from infectious and
non-infectious diseases of the lungs are found to be more risk from this viral infection due to the lower
immune system[1]. Hence, enhancing the immunity (natural body system) may possess the major
contribution as a prophylactic measure against multiple pathogenic conditions as well as maintaining
optimum health [2].

Ayurveda utilizes the concept of “Dinacharya” and “Ritucharya” to maintain healthy life that utilizes the
gifts of nature (herbal medicines) as daily/seasonal regimes to maintain a healthy life [3]. Ayurveda; a
plant-based science suggests in simplifying the lifestyle, and also promotes the awareness in uplifting
and maintaining own’s immune system via the utilization of many plants/herbs [3] which are easily
available at the kitchen garden of a majority of the society.

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Based on the Ayurvedic and scienti c literature, Ministry of AYUSH (Ayurveda, Yoga and Naturopathy,
Unani, Siddha, and Homeopathy), India issued advisory where it recommended the use of Kadha (herbal
tea/decoction) composed of Ocimum tenui orum (Tulsi), Cinnamomum verum  (Dalchini), Piper nigrum 
(Kalimirch), Zingiber o cinale  (Shunthi) and Vitis vinifera (Munakka) for self-care which will develop
immunity against severe infection caused by COVID-19. Further, it also recommended the consumption of
golden milk; a half teaspoon of Curcuma longa (turmeric) powder in 150 ml hot milk once or twice a day
[3]. However, the scienti c evidence of all these herbal combinations for boosting the immune system has
not been proposed yet. Hence, in the present study, we proposed to elucidate the probable interaction of
the phytoconstituents from individual ingredients of the AYUSH recommended formulation to boost the
immune system by gene-set enrichment and network pharmacology approach to support with scienti c
shreds of evidence.

2. Materials And Methods

2.1 Mining of phytoconstituents and their targets

The phytoconstituents of 6 medicinal plants i.e. Cinnamomum verum, Curcuma longa, Ocimum
tenui orum, Piper nigrum, Vitis Vinifera, Zingiber o cinale were retrieved from ChEBI
(https://www.ebi.ac.uk/chebi/) database. The targets of each phytoconstituents were identi ed using
DIGEP-Pred [4] for proteins at Probable activity (Pa)>0.7.

2.2 Enrichment and Network analysis

The list of probable targets was queried in STRING [5] database, and enrichment analysis of protein-
protein interaction was performed for biological process, molecular function, and cellular components.
Further, the probably modulated pathways were also identi ed concerning the KEGG pathway database.
Cytoscape [6] was used to construct the network among the plants, their phytoconstituents, modulated
proteins, and regulated pathways. The network was treated as directed, node size as “low values to small
sizes” and map node color from “low values to bright colors” and the whole network was analyzed based
on edge count.

2.3 Druglikeness and ADMET Pro le

Druglikeness score of each phytoconstituent was calculated based on Lipinski’s rule of ve [7] using
MolSoft (https://molsoft.com/mprop/). Likewise, the probability for human intestinal absorption, caco-2,
and blood-brain barrier permeability, human oral bioavailability, P-glycoprotein, CYP3A4, CYP2C9,
CYP2D6, and CYP1A2 inhibition, eye irritation, Ames mutagenesis, human ether-a-go-go inhibition, and
hepatotoxicity was predicted using admetSAR2.0 [8].

3. Results
3.1 Phytoconstituents and their targets

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Total 221 phytoconstituents were identi ed in which 40 were from Cinnamomum verum, 27 from
Curcuma longa, 3 from Ocimum tenui orum, 91 from Piper nigrum, 40 from Vitis vinifera, and 20 from
Zingiber o cinale in which 173 compounds were identi ed to modulate the proteins at Pa>0.7. Among
them, (-)-(3S)-1-(3,4-hydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol was predicted to have the highest
interaction with multiple proteins i.e. 18. Likewise, MMP2 was predicted to be a majorly targeted protein
by 58 phytoconstituents. The list of phytoconstituents and their source of each compound are
summarized in Table S1. By these 173 phytoconstituents, a total of 524 interactions were made with
multiple proteins. The complete interaction of the multiple phytoconstituents with their targets and
regulated pathways is represented in Fig. 1.

3.2 Gene set enrichment analysis

Gene set enrichment analysis identi ed eighty different pathways to be modulated by the bioactives from
the herbal tea. Phytoconstituents were also identi ed to regulate the proteins which were involved in
multiple pathogeneses (infectious and non-infectious diseases) including immunology (Table S2). The
protein-protein interaction of each modulated proteins and their gene GO analysis is represented in Fig. 2
and Fig. 3 respectively.

3.3 Druglikeness and ADMET pro le

Among 221 phytoconstituents, eighty compounds were predicted to possess a positive druglikeness
score in which camphoratin D from Cinnamomum verum scored highest druglikeness score i.e. 1.18 (Fig.
4). Among the 20 best hits from the list of phytoconstituents, all the compounds were predicted to get
absorbed from the human intestinal tract. Among them, camphoratin B was predicted to have the highest
oral bioavailability and was also predicted for the least toxicity pro le like eye irritation, Ames
mutagenesis, human either-a-go-go inhibition, and hepatotoxicity compared to rest of compounds (Fig.
5).

4. Discussion
Recently, AYUSH has advised utilizing Kadha (herbal tea/decoction) composing basil, cinnamon, black
pepper, dry ginger, and raisin and golden milk to boost the immune system as a prophylactic measure
against COVID-19. Due to the complex composition of multiple phytoconstituents from all these
medicinal plants, the mixture of this combination could modulate multiple proteins and would help to
boost the immune system which can be explained via network pharmacology and gene-set enrichment
analysis. Network pharmacology and gene set enrichment analysis are well-accepted approaches to
identify the disease target, lead hit molecules and modulated pathways via “multiple component-protein
interactions” [9-11].

Boosting of the immune system involves the modulation of multiple proteins that are involved in the
homeostatic regulation. In the present analysis, we identi ed the modulation of multiple pathways that
are related to infectious/non-infectious diseases and the immune system. A subject suffering from
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infectious/non-infectious diseases possesses a lower immune system [12]; are at higher risk to be
affected by COVID-19. Hence, the herbal tea which has been recommended by the AYUSH is not limited
over the boosting of the immune system but also may modulate other pathways which are involved in the
pathogenesis of multiple diseases provides bene cial effect to the patients with diabetes and
hypertension who cannot undergo to the pharmacotherapy of 4-hydroxy quinoline.

Enrichment analysis of modulated proteins identi ed the regulation of HIF-1, p53, PI3K-Akt, MAPK, cAMP,
Ras, Wnt, NF-kappa B, IL-17, TNF, and cGMP-PKG signaling pathway which are directly involved in
boosting the immune system. The task of HIF-1α has been reported to be dysregulated in viral infection
which is involved to boost the immune system by regulating the task of macrophages, neutrophils,
dendritic cells, and lymphocytes during the hypoxic condition [13]; primarily occurs in COVID-19 infection
due to improper exchange O2/CO2 in lungs [14]. In the present study, we identi ed ve genes i.e. ALDOA,
FLT1, HMOX1, NOS2, and TIMP1 to be modulated related to this pathway. Further, PI3K-Akt functions as a
rheostat in orchestrating the differentiation of memory CD8 T cells [15] which further regulate the
functioning of multiple chemokines and cytokines [16] from multiple pathways like NF-kappa B, IL-17,
and TNF signaling pathway which were found to be modulated in the present study. Hence, the next
approach to boost the immune system by suggested Kadha could occur via regulation of PI3K-Akt, NF-kβ,
interleukin, and TNF mediated cytokine regulation. Further in infectious diseases including viral infection
MAPK pathway gets targeted by pathogens. Since this pathway is involved in the synthesis of
immunomodulatory cytokines like interleukins and TNF-α via the activation of p38 MAPK pathways,
modulation of this pathway could play important coordination with an immune response via the
promotion of Th1 and Th2 against extracellular infectious agents [17]; has been regulated in the present
study via the modulation of six proteins i.e. CD14, FLT1, HSPA6, RAC1, RAP1A, TNFRSF1A. Further, in
COVID-19 there is an increase in cell apoptosis and necrosis in lung tissue. As the phytoconstituents were
also predicted to modulate the Ras signaling pathway, the suggested herbal tea could also function in
multiple cellular tasks including the regulation of cell survival and proliferation [18]. Additionally, we also
identi ed the multiple pathways (Table S2) which get modulated in the pathogenesis of multiple
infectious (bacterial/viral) and non-infectious diseases (diabetes, obesity, hypertension, etc) in which the
immune system is compromised. Hence, the intake of this tea would also add a bene cial effect to
maintain their daily lifestyle and also may improve the immunity system.

Since the AYUSH suggested the intake of the composition as an herbal tea and is to be taken orally, we
attempted to identify the probable lead hits to get absorbed from the human intestinal tract in which the
majority of the compounds were predicted to be absorbed from the gastrointestinal tract. Based on the
Rule of Five, we identi ed camphoratin D to possess the highest drug-likeness score. Similarly,
camphoratin B was predicted to have the highest human oral bioavailability. However, the single-molecule
may not be as effective compared to the advised herbal tea as the amount required to boost the immune
system may not su cient enough. Hence, intake of the multiple compounds in the form of herbal tea as
suggested by AYUSH could be more bene cial rather than a single molecule for boosting the immune

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system as a prophylactic against COVID-19 and could be helpful in subjects who are  suffering diabetes
and hypertension in which 4-hydroxychloroquine treatment cannot be proceed.

5. Conclusion
The present study utilized the system biology tools to assess the immunomodulatory effect of Kadha
(herbal tea) suggested by AYUSH as a prophylactic approach against COVID-19. In this study, we
identi ed the suggested herbal tea may not be limited to enhance the immune system but also may
regulate multiple proteins/pathways involved in the infectious and non-infectious diseases which could
be bene cial to those subjects in which 4-hydroxy quinoline cannot be administered.

Declarations
Con ict of interest

The authors declare that they have no con ict of interest.

Foundation project

The authors have no support or funding to report.

Acknowledgment

Authors are thankful to Principal KLE College of Pharmacy, Belagavi, KLE Academy of Higher Education
and Research (KAHER) Belagavi for his support.

Authors Contribution

Yadu Nandan Dey made the concept and designed the study. Pukar Khanal reviewed literature, carried out
the work and drafted the manuscript. Taaza Duyu assisted in the mining of data and drafting/reviewing
the manuscript. BM Patil and Yadu Nandan Dey have contributed to suggesting re ning the protocol and
nal draft of the manuscript. Ismail Pasha contributed to the nal re ning of the manuscript.

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Figures

Figure 1

Interaction of phytoconstituents with their proteins and regulated pathways

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Figure 2

Protein-protein interaction of regulated proteins by the phytoconstituents from herbal tea

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Figure 3

GO gene analysis of modulated proteins (a) cellular components (b) molecular function and (c) biological
process

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Figure 4

Druglikeness score of phytoconstituents. MW: Molecular weight, NHBA: Number of hydrogen bond
acceptor, NHBD: Number of hydrogen bond donor, BBB S: Blood brain barrier Score, NSC: Number of
stereo centres, DLS: Druglikeness score

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Figure 5

Fig. 5:cinnamtannin B-1, (b) cinnamtannin D-1, (c) cassiatannin A, (d) parameritannin A-1, (e) procyanidin
B2, (f) cinnamtannin A2, (g) zhankuic acid B, (h) zhankuic acid C, (i) zhankuic acid A methyl ester, (j)
Camphoratin A, (rel)-, (k) Camphoratin B, (l) Camphoratin C, (m) Camphoratin D, (n) Camphoratin E, (o)
Camphoratin F, (p) Camphoratin J, (q) antcin A, (r) antcin C, (s) methyl 4-α-methylergost-8,24(28)-diene-
3,11-dion-26-oate and (t) procyanidin B1

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