Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183

Contents lists available at ScienceDirect

Pharmacological Research - Modern Chinese Medicine

journal homepage: www.elsevier.com/locate/prmcm

Network pharmacology-based elucidation of bioactive compounds in

propolis and putative underlying mechanisms against type-2 diabetes
mellitus✰
Emmanuel I. Ugwor a,b,∗, Adewale S. James c, Adekunle I. Amuzat b, Emmanuel O. Ezenandu b,
Victory C. Ugbaja b, Regina N. Ugbaja b
a
Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
b
Department of Biochemistry, College of Biosciences, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria
c
Department of Chemical Sciences, Faculty of Science, Augustine University, Ilara-Epe, Lagos State, Nigeria

a r t i c l e i n f o a b s t r a c t

Keywords: Propolis is a common remedy in Chinese medicine. We have previously demonstrated the anti-diabetic poten-
Nigerian propolis tials of Nigerian propolis. However, the underlying mechanisms against type-2 diabetes mellitus (T2DM) remain
Network pharmacology unclear. This study used network pharmacology-based analysis to unravel the possible mechanisms of NP’s anti-
Type-2 diabetes
T2DM activity. Previously identified compounds in NP were retrieved from literature, screened for drug-likeness,
Bioactive compounds
and used to retrieve targets associated with T2DM, from which compound-target, protein-protein interaction,
and target-pathways networks were constructed. Network pharmacology-based and enrichment analyses were
conducted on the networks. Further, NP’s inhibitory activity against targets identified in network analyses was
assessed in-vitro. Library search revealed 202 previously reported compounds in NP; 96 were retained after
screening for drug-likeness. However, only 44 NP compounds interacted with T2DM-associated targets, with
2-ethyl-1,4-dimethoxybenzene as the most active component. Network analyses revealed 167 putative targets,
with HSP90AA1, JUN, ESR1, STAT3, CYP3A4, PTGS2, RELA, VEGFA, CYP2C9, and PPARG as the core anti-T2DM
targets of NP compounds. Gene ontology analyses indicated that these targets were predominantly localised in
the plasma membrane and cytoplasm and primarily involved in regulatory, signal transduction and cellular re-
sponse processes. KEGG pathway enrichment implicated metabolic pathways (involving lipids), AGE-RAGE, and
calcium/cAMP, among others, in the anti-T2DM effects of the compounds. Furthermore, in vitro pharmacologi-
cal assessment demonstrated appreciable inhibitory effects of 2-ethyl-1,4-dimethoxybenzene against 𝛼-amylase,
𝛼-glucosidase, and HMG-CoA reductase. This study provides holistic mechanistic insights into the anti-T2DM
activities of the constituents of Nigerian propolis.

1. Introduction
Type-2 diabetes mellitus (T2DM) is one of the four major non-
communicable diseases that has continually afflicted the global popu-
lace and has been hallmarked as the epidemic of the millennium [1,2].
Globally, the prevalence of T2DM has increased by 49% since 1990. In
2019, there were 437.9 million reported cases of T2DM, with the highest
proportion in low- and middle-income countries [3]. T2DM is a chronic
metabolic condition brought on by impaired insulin production by pan-
creatic beta cells and insulin resistance in peripheral insulin-sensitive
organs [4]. The aetiology of T2DM stems from various environmental,
genetic and epigenetic factors and is associated with several metabolic
diseases, such as obesity and overweight. It is also a risk factor for heart
disease – the leading cause of death globally [5].
Although T2DM prognosis has improved and several anti-T2DM ther-
apies have been formulated over the years, its prevalence continues to
surge [6]. Pharmacological agents, such as sulfonylureas, glucagon-like
peptide1 agonists, HMG-CoA reductase inhibitors, and 𝛼-glucosidase
inhibitors, and lifestyle modification have been the mainstream anti-
T2DM therapies [6]. However, these agents are limited by adverse side
effects and high costs. Besides, they often target one aspect of the other-
wise multifaceted complications associated with T2DM [7]. Conversely,
natural products, encompassing herbal remedies, possess few side ef-
fects and have increasingly been exploited by researchers in developing
therapeutic agents against T2DM [8].

✰
Type of Paper: Original article.
∗
Corresponding author at: Department of Biochemistry, College of Biosciences, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria.
E-mail address: ugworei@funaab.edu.ng (E.I. Ugwor).

https://doi.org/10.1016/j.prmcm.2022.100183
Received 28 August 2022; Received in revised form 12 October 2022; Accepted 20 October 2022
Available online 21 October 2022
2667-1425/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
E.I. Ugwor, A.S. James, A.I. Amuzat et al.
Propolis is one of the natural products investigated for its efficacy
against T2DM [9–12] and is a common remedy in Chinese medicine
[9]. It is a resinous substance produced by honey bees from mixing their
salivary and enzymatic secretions with plant sap, buds, gums, and other
vegetal materials [13]. Propolis is abundant in bioactive compounds,
such as flavonoids, terpenes, aromatic acids and their esters, which en-
dow it with an extensive range of biological properties, including an-
tioxidant, antibacterial, anti-inflammatory, anti-diabetic and anticancer
properties [11,12]. However, the chemical constituent of propolis is
highly divergent, depending heavily on the bee species, the flora sur-
rounding the collection site, and geographical zones [13]. In Nigeria,
propolis has purportedly been used to treat ringworm, chickenpox, and
measles and manage diabetes [12]. We have previously demonstrated
the anti-diabetic potentials of Nigerian propolis (NP), including hypo-
glycemic, hypolipidemic, and hepatoprotective properties, in rats with
streptozotocin-induced diabetes [13]. Other studies have reported sim-
ilar anti-diabetic properties of NP [10,12,15]. However, the underlying
mechanisms of NP anti-T2DM effects have not been elucidated.
The varied bioactive components in natural products and their po-
tential to act synergistically on multiple targets pose limitations to elu-
cidating their mechanisms of action [16]. However, recent advances in
computer-aided drug designs, such as network pharmacology, now al-
low a holistic view of the multicomponent-multi target paradigm of nat-
ural products, thus revealing the putative mechanisms of action against
different diseases via interacting targets and associated pathways [17–
19]. Network pharmacology is a scope in systems biology that visualises
the interactions of the bioactive compound(s) with both the interactome
and the diseasome, thus heightening the understanding of the multi-
target mechanism of the phytochemical constituents of natural products
via a compound–target–pathway-disease network constructs [16,19].
Therefore, this study utilised a network-based pharmacology anal-
ysis to unravel the possible mechanisms of NP anti-T2DM activity and
identify responsible bioactive components. In addition, the inhibitory
activity of NP’s bioactive component against some targets identified was
also evaluated.
2. Methods
2.1. Assembling of compounds database for Nigerian propolis
A database of ligands in Nigerian propolis was compiled from pre-
viously reported works conducted on the characterisation of propolis
within Nigeria. The canonical SMILES of each compound were retrieved
from PubChem (https://pubchem.ncbi.nlm.nih.gov/). One compound
(riverinol) had no deposition in PubChem, so its SMILES was deduced
from its structure using Marvin JS plugin in SwissADME online server
(http://www.swissadme.ch/index.php).
2.2. Screening of compounds
Compounds were screened based on their absorption, distribution,
metabolism and excretion (ADME) properties, bioavailability score, and
violations of the rules of the Lipinski et al. [20], Veber et al. [21], and
Ghose et al. [22] drug-likeness approaches, which was computed on
the SwissADME server. Compounds with two or more violations and
estimated oral bioavailability < 50% were excluded from further analy-
ses. In addition, 37 FDA-approved drugs for T2DM were from DrugBank
(https://go.drugbank.com), and their pharmacological properties were
computed with SwissADME (Supplementary File 1). Principal compo-
nent analysis (PCA) was conducted on the calculated properties [17];
the two most significant principal components were used to compare
the chemical space between the screened compounds and drugs in a
scatterplot.
2
Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183
2.3. Compounds- and disease-associated targets
On the one hand, the SMILES of the screened compounds
were used to retrieve linked target genes from the Similarity
Ensemble Approach (SEA; http://sea.bkslab.org/) and (Swiss Tar-
get Prediction (STP; http://www.swisstargetprediction.ch/) databases
[23]. On the other hand, using the Homo sapiens mode and
“type 2 diabetes mellitus” as the keyword, T2DM-linked targets
were recovered from the DisGeNeT (https://www.disgenet.org/),
Malacards (https://www.malacards.org/), and Kyoto Encyclopedia
of Genes and Genomes (KEGG; https://www.kegg.jp) databases
[18]. Subsequently, overlapping targets between the compound-
associated and ND-associated targets were identified using VENNY 2.1
(https://bioinfogp.cnb.csic.es/tools/venny/) and used for network con-
struction.
2.4. Network construction and analyses
Three networks – compound-target (CT), protein-protein interaction
(PPI), and target-pathways (TP) networks) – were constructed using Cy-
toscape 3.9.1 software (http://www.cytoscape.org/) to visualise the in-
teraction between the screened bioactive compounds, selected targets,
and associated pathways [24]. First, the screened compounds and the
associated overlapping targets were used to construct the CT network.
Next, the overlapping targets were uploaded onto the STRING database
(Version 11.5; (https://string-db.org/) to predict the PPI [25]. Protein
interactions with confidence level > 0.7 within the Homo sapiens mode
were then used to construct the PPI network in Cytoscape. Core com-
pounds and targets were identified using the degree algorithm of the
CytoHubba plugin in the Cytoscape to analyse the CT and PPI networks,
respectively. Further, the nodes in the PPI network were aggregated into
clusters using the MCODE plugin within Cytoscape with the following
parameters: Degree cutoff = 2, Node Score Cutoff = 0.2, and K-Core = 2.
Information about each cluster was fetched directly from the STRING
database using its application programming interface domiciled in Cy-
toscape.
The overlapping targets were mapped onto the Comparative Tox-
icogenomics Database (CTD; http://ctdbase.org/tools/) to extract the
canonical pathways highly associated with these proteins. Within CTD,
gene inputs identify pathways involving these genes based on annota-
tion from the KEGG and REACTOME databases. Non-specific and non-
diabetes-related terms were removed, and the TP network was con-
structed in Cytoscape to present a global view of the interactions be-
tween targets and associated pathways.
2.5. Gene ontology (GO) and KEGG pathway enrichment analyses
The Database for Annotation, Visualization and Integrated Discovery
(DAVID; https://david.ncifcrf.gov/tools.jsp) server was used for enrich-
ment analyses [16]. The overlapping genes were uploaded onto DAVID,
the identifier was OFFICIAL GENE SYMBOL, species was set as Homo
sapiens, and information on the cellular component (CC), biological pro-
cess (BP), molecular function (MF), and KEGG pathways pertinent to
each target were retrieved. Only GO terms and KEGG pathways with
a p-value of less than 0.05 and Benjamini value of less than 0.5 were
retrieved. Within the DAVID server, the p-values were adjusted for the
false discovery rate (i.e., Q-values) [23]. GraphPad Prism 9.3.1 was used
to visualise the top ten most enriched components of the GO and KEGG
enrichment analyses.
2.6. In-vitro pharmacological validation
The anti-diabetic potentials 2-ethyl-1,4-dimethoxybenzene (identi-
fied from network analyses as the most active NP component) were
evaluated by assaying for the inhibitory activity against selected targets
E.I. Ugwor, A.S. James, A.I. Amuzat et al.
identified by network analyses: 𝛼-amylase, 𝛼-glucosidase, and HMG-
CoA reductase (HMGR). While 2-ethyl-1,4-dimethoxybenzene (Cat. NO.:
3452AA) was procured from Ak Scientific, Inc. (Union City, CA, USA),
the enzymes and other reagents used for the in-vitro assays were prod-
ucts of Sigma-Aldrich (St. Louis, MO, USA).
2.6.1. Anti-diabetic activity
Briefly, 100 𝜇L of the different concentrations (20, 40, 60, 80,
100 𝜇g/ml) of 2-ethyl-1,4-dimethoxybenzene or acarbose (Cat. NO.:
A8980) and 500 𝜇l of 20 mM sodium phosphate buffer (pH 6.9) contain-
ing 6 mM NaCl and pancreatic 𝛼-amylase (10 U/ml; Cat. No.: A3176)
were incubated at 25 °C for 10 min. The reaction was initiated by adding
1% starch solution (500 𝜇l, prepared in the same phosphate buffer) and
monitored at 540 nm for 10 min [26].
For the 𝛼-glucosidase assay, 100 𝜇L of the 𝛼-glucosidase solution
(Cat. No.: G5003) was allowed to equilibrate with 50 𝜇L of 2-ethyl-1,4-
dimethoxybenzene or acarbose (20, 40, 60, 80, 100 𝜇g/ml) at 25 °C for
10 min. Then, 50 𝜇L of 5 M p-nitrophenyl-𝛼-D-glucopyranoside solution
(in 0.1 M phosphate buffer; pH 6.9) was added, and the reaction was
monitored at 405 nm for 5 min [27].
For HMGR, assay kit (Sigma; Cat. No.: CS1090) containing the cat-
alytic domain of the human enzyme and other reagents was used for
the HMGR inhibitory assay, as previously described [28]. Briefly, 20 𝜇L
of the enzyme (0.5 mg/ml) and 10 𝜇L of 2-ethyl-1,4-dimethoxybenzene
or simvastatin (Cat. NO.: S6196) were added to the reaction mixture
containing 400 𝜇M NADPH and 400 𝜇M HMG-CoA substrate in a final
volume of 2 mL of 100 mM potassium phosphate buffer, pH 7.4 (con-
taining 120 mM KCl, 1 mM EDTA, and 5 mM DTT). The rate of NADPH
disappearance was monitored at 340 nm for 15 min. The blanks con-
tained neither 2-ethyl-1,4-dimethoxybenzene nor reference drugs.
Tests were performed in triplicates, and the percentage inhibition for
each enzyme was calculated using the formula below:
Δabsorbence blank − Δabsorbence sample
%Inhibition = × 100
Δabsorbence blank
2.7. Data management
Data retrieved or obtained in this study were managed with Mi-
crosoft Excel (2019). Graphs were graphs using GraphPad Prism 9.3.1
(GraphPad Software Inc., CA, USA). GraphPad was used to fit results ob-
tained from the in-vitro analyses into a dose-response curve to determine
IC50 values.
3. Results and discussion
3.1. Nigerian propolis compound database
A literature search revealed five previous original researches that
characterised the phytochemical composition of Nigerian propolis
[12,29–32], from which we extracted 202 compounds, compiled in Sup-
plementary File 1. The compounds were then screened using the ADME
properties, three drug-likeness approaches, and projected oral bioavail-
ability. A compound’s ADME features predict its disposition inside an
organism, contributing to its pharmacological (or toxicological) action
[33]. The oral bioavailability indicates the fraction of the compound
that will reach systemic circulation unchanged following oral admin-
istration, which correlates with its efficacy. Furthermore, the rules of
Lipinski, Ghose, and Verber are drug-likeness approaches used to iden-
tify whether a chemical molecule with a particular pharmacological or
biological activity has chemical and physical qualities, such as molecu-
lar weight, partition coefficient, and the number of hydrogen donors and
acceptors, which would make it a potential orally active drug [20–22].
Only compounds meeting these criteria were used for further analyses.
Ninety-six compounds were included (Supplementary File 2), while 106
compounds violated these criteria and were excluded.
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Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183
Fig. 1. The distribution in chemical space according to principal component
analysis of screened compounds in Nigerian propolis (n = 96) and FDA-approved
drugs (n = 37). The two most significant principal components (PC1 and PC2)
were used to compare the chemical space.
The chemical space occupied by these screened compounds was then
compared to those of FDA-approved drugs using the two most signifi-
cant principal components, which explained 53.0% and 26.0% of vari-
ances, respectively (Fig. 1). Although both groups occupied two distinct
clusters, there were clear overlaps between the two groups, further il-
lustrating the drug-like potentials of the compounds.
3.2. Elucidation of T2DM-associated targets of screened Nigeria propolis
compounds
Network pharmacology-based analysis was used to identify T2DM-
associated targets of the screened compounds. Targets associated with
each compound were retrieved from SEA (580 targets and STP (927
targets), while T2DM-associated targets (n = 3395) were retrieved from
DisGeNeT, Malacards, and KEGG databases. Comparing these three data
sets, we uncovered 167 overlapping targets (Fig. 2) common to both
compounds- and disease-associated targets (detailed in Supplementary
File 3).
These common targets were then entered into the STRING database
to analyse the protein-protein interactions, which comprise direct (phys-
ical) and indirect (functional) associations, obtained from computa-
tional prediction, inter-organismal knowledge transfer, and associations
culled from other databases [25]. The significant interactions (with a
high confidence level of >0.7) were visualised as PPI-network (Fig. 3A).
The PPI network included 135 nodes connected by 371 edges after elim-
inating isolated nodes and edges, with an average node degree of 4.49.
The substantial number of interactions between the targets suggests that
they may be biologically related, and the anti-T2DM effects of the com-
pounds may involve multiple targets. The top 10 targets (hereafter re-
ferred to as core targets) in the PPI network were identified based on
degrees, betweenness, and closeness parameters, following the analysis
of the network with the CytoHubba algorithm (Supplementary File 4).
The core targets were HSP90AA1, JUN, ESR1, STAT3, CYP3A4, PTGS2,
RELA, VEGFA, CYP2C9, and PPARG (Fig. 3B); these targets accounted
for 47.8% of the total protein-protein interactions (167 of 371 PPI). Be-
sides, several studies have highlighted the importance of these targets
to T2DM. Lazaro et al. [34] reported that pharmacological inhibition of
HSP90 ameliorated diabetes-associated complications, while Yang et al.
[35] revealed decreased Hsp90 expression in isolated rat islets exposed
to high glucose. They surmised that Hsp90 might be involved in high
glucose-induced islet dysfunction. Meanwhile, the cross-talk between
JUN, RELA, and STAT3 has been implicated in the progression of T2DM
[36,37]. Clinical and pre-clinical studies have reported the roles of CYP,
PTGS2, and VEGFA in diabetes [38–40]. ESR1 mediates the effects of es-
trogens on glucose homeostasis [41].
E.I. Ugwor, A.S. James, A.I. Amuzat et al.
Fig. 2. Venn diagram illustrating the overlapping targets (central) between
compound-associated disease targets (CAT) obtained from Similarity Ensemble
Approach (SEA) and Swiss Target Prediction (STP) databases, as well as the
disease-associated targets (DAT).
To provide more mechanistic insights, we clustered the PPI-network
nodes using the MCODE plugin in Cytoscape (Fig. 4); information about
each cluster was fetched directly from the STRING database. Nodes
in cluster 1 were involved in lipids (phospholipids arachidonate, and
Fig. 3. Protein-protein interaction network (A.) and the top ten (core) type-2 diabetes disease targets (B.) associated with screened compounds in Nigerian propolis.
Core targets were identified based on the number of degrees, betweenness, and closeness using the degree algorithm of the CytoHubba plugin within Cytoscape
(version 3.9.1).
4
Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183
eicosanoids) metabolism; cluster 2 nodes are primarily involved in cellu-
lar responses to stimuli, such as hypoxia (HIF-1), inflammation (NFKB1,
STAT3, and RELA), heat (HSP90), proliferation (VEGF, PDGFRB, PRKCZ
and PPARG), and tissue remodelling (MMPs and VEGFA). Clusters 3 and
4 were involved in signal transduction and alcohol metabolism, respec-
tively. Nodes in cluster 5 have been correlated to amyloid deposition,
while those in clusters 6 and 7 regulate adenylate cyclase/cGMP down-
stream signalling and neurotransmitter transport/activity, respectively.
Nodes in cluster 8 share phosphor-amino acids (e.g., serine, threonine,
or tyrosine) motifs and function as adapter proteins.
Interestingly, of the 96 compounds, only 44 had targets common to
T2DM-associated targets (Table 1), whereas 52 compounds had no over-
lap with T2DM targets. The interactions between overlapping targets
and the screened compounds were visualised in a CT network (Fig. 5A),
consisting of 221 nodes (44 compounds and 167 targets) and 620 inter-
actions, with an average of 3.52 targets/compound. The overlap of tar-
gets reflects the multi-target characteristics of the bioactive compounds.
The CT network was subsequently analysed with CytoHubba to identify
hit compounds (Supplementary File 4). The top 10 compounds, based
on degree, are presented in Fig. 5B. The most active compound – 2-
ethyl-1,4-dimethoxybenzene – interacted with 63 targets, followed by
borneol (32). These top compounds accounted for 73.2% of compound-
target interactions (227 of 310).
The overlapping targets were then linked to biochemical pathways
using the CTD online server; the highly significant T2DM-linked path-
ways were visualised as a network with the targets and associated path-
ways represented as nodes and the associations as edges (Fig. 6). In this
way, the 143 targets were mapped to 43 canonical pathways, with 576
interactions between these components, further highlighting the diverse
mechanisms at play in propolis anti-T2DM activity.
Next, the overlapping targets were annotated using DAVID and re-
trieved three GO terms – CC, BP, and MF (Supplementary File 5). The top
10 terms for each category are presented in Fig. 7. Almost half (49.4%)
of the targets can be found in the plasma membrane; 42.2, 38, and 29.5%
were within the cytoplasm, integral component of the membrane, and
E.I. Ugwor, A.S. James, A.I. Amuzat et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183

Fig. 4. Cluster analysis of the targets in the protein-protein interaction network. Cluster analysis was performed with the MCODE plugin within Cytoscape (version
3.9.1) with the following parameters: Degree cutoff = 2, Node Score Cutoff = 0.2, and K-Core = 2.

Fig. 5. Compound-target network (A.) and top ten compounds in Nigerian propolis (B.) against overlapping type-2 diabetes disease targets. Compounds are ranked
for bioactivity based on the number of degrees, betweenness, and closeness using the degree algorithm of the CytoHubba plugin within Cytoscape (version 3.9.1).
Green nodes = compounds, blue nodes = targets.

5
 E.I. Ugwor, A.S. James, A.I. Amuzat et al.
Table 1
Bioactive compounds in Nigerian propolis against type-2 diabetes mellitus, as identified by network pharmacology analysis.

Rank Compound Parameters∗ Rank Compound Parameters∗

Degree Betweenness Closeness Degree Betweenness Closeness

1 2-Ethyl-1,4-dimethoxybenzene 63 22,890.92 105.53 23 Gerontoxanthone H 2 647.62 69.66

2 Borneol 32 8453.39 80.53 24 Vesticarpan 2 334.41 71.17
3 3,8-dihydroxy-9-methoxy-pterocarpan 27 7772.28 79.23 25 Calycosin 2 67.46 54.95
4 Kaempferol 25 5349.30 72.02 26 6-Prenylnaringenin 2 56.79 51.70
5 Decanoic acid, methyl ester 24 6478.51 76.83 27 Octanoic acid 1 0.00 64.96
6 Resveratol 15 4971.80 70.78 28 a-Copaene 1 0.00 53.89
7 13-Tetradece-11-yn-1-ol 13 2576.47 63.52 29 a-melonol 1 0.00 53.89
8 Broussonin B 10 2469.24 62.07 30 cis-Geraniol 1 0.00 53.89
9 1,5,9,9-tetramethyl-1,4,7-Cycloundecatriene 9 2062.24 61.59 31 Vestitol 1 0.00 53.89
6

10 2-(7-Octenyl)oxirane 9 1824.00 63.34 32 Quinine 1 0.00 46.30

11 4-epi-a-Acoradiene 8 1577.66 55.85 33 Ribalinidine 1 0.00 46.30
12 Dodecanoic acid 7 1566.00 57.46 34 Linalool oxide 1 0.00 45.88
13 Pterocarpan 6 1825.46 55.89 35 Cyclosativene 1 0.00 44.30
14 Lunamarin 6 1737.42 57.18 36 Caryophyllene oxide 1 0.00 43.51
15 Catechin 5 1048.04 51.43 37 Methyl chavicol 1 0.00 42.72
16 4,4-Dipropylheptane 5 127.81 50.29 38 Medicarpin 1 0.00 39.30
17 Benzene, 1-methyl-4-(2-pentenyloxy) 4 989.00 45.42 39 Citral 1 0.00 35.88

Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183

18 Naringenin 4 608.45 55.59 40 Flavan-3-ol 1 0.00 34.30
19 Pinocembrin 4 6.00 50.57 41 Humulene-1,2-epoxide 1 0.00 30.86
20 a-Calacorene 3 689.90 55.28 42 Dodecane 1 0.00 1.50
21 Epihedrine 3 252.37 54.36 43 Ethyl 2-(5-methyl-5-vinyltetrahydrofuran-2-yl) propan-2-yl carbonate 1 0.00 1.00
22 b-Methylnaphthalene 2 1188.00 54.94 44 Heptane, 2,4,6-trimethyl- 1 0.00 1.00
∗
Parameters were calculated from analysing the compound-target network with the CytoHubba in Cytoscape (version 3.9.1).
E.I. Ugwor, A.S. James, A.I. Amuzat et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183

Fig. 6. Target-pathway network. Dark green nodes = path-

way, blue nodes = targets.

Fig. 7. Top 10 components of Gene Ontology

(GO) enrichment analyses of Nigerian propo-
lis compounds’ anti- type-2 diabetes disease
targets. (A) Cellular component; (B) Biological
process; and (C) Molecular function.

7
E.I. Ugwor, A.S. James, A.I. Amuzat et al.
Fig. 8. Bubble plot showing top 10 highly enriched pathways obtained from
Kyoto encyclopaedia of Genes and Genomes (KEGG) pathway assessment.
NLRI = neuroactive ligand-receptor interaction.
nucleoplasm, while 14.5% can be found in the mitochondrion. Regula-
tory, signal transduction and cellular response processes accounted for
the top 10 biological processes. These processes are closely related. For
example, external/internal stimuli (e.g., insulin or low glucose levels)
can act as molecular signals, which the cell amplifies or transmits, usu-
ally involving signalling cascades of protein interactions, and leads to
the regulation of several aspects of the biological system, in response to
the stimuli.
Furthermore, BP annotation indicated that 18.1 and 10.8% of the
overlapping targets were involved in the positive regulation of transcrip-
tion and apoptosis, while 10.8% and 12.7% were negative regulators of
these processes, respectively. Meanwhile, 16.9 and 13.3% were involved
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Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183
in G-protein coupled receptor (GPCR) signalling and signal transduc-
tion, while 12.7% and 12.0% were associated with inflammatory and
drug response, respectively. These biological processes correspond with
MP annotation, revealing that 75.3% of the targets’ functions involved
protein binding, a central regulatory and signalling mechanism. Other
functions include binding to DNA, zinc, and ATP, protein homodimerisa-
tion, kinase, and GPCR activities, all of which are involved in signalling
and regulation. Indeed, T2DM develops from dysfunctional insulin sig-
nalling and has been associated with impaired regulatory and signalling
mechanisms [42,43].
KEGG pathways enriched for the overlapping targets were retrieved
from the DAVID online tool; pathways not associated with T2DM (e.g.,
chemical carcinogenesis) or not having P-values less than 0.05 were ex-
cluded (Supplementary File 5). The ten most enriched pathways are pre-
sented in a bubble plot against the fold enrichment (Fig. 8). 31.9% of
the targets were involved in metabolic pathways, which is unsurprising
given that T2DM is a metabolic disease [43]. Differentially expressed
genes linked to neuroactive ligand-receptor interaction (12.7% of tar-
gets) have been reported in diabetic patients [44,45]. Advanced glyca-
tion end products (AGEs) interact with their receptor (RAGE) to potenti-
ate crosstalk that amplifies T2DM-associated complications [36]. In con-
trast, cAMP/adenylate cyclase/calcium crosstalk has been posited as a
pharmacological target to enhance 𝛽-cell survival/function and improve
glucose homeostasis [46]. Ras and MAPK8 (c-Jun N-terminal kinase)
signalling have been implicated in the orchestration of intricate events
that lead to chronic low-grade inflammation characteristic of T2DM and
its microvascular complications [47]. The PI3K-Akt pathway is crucial
in mediating insulin’s metabolic effects. Insulin binds to its receptor,
which phosphorylates insulin receptor substrates, promoting the activa-
tion of PI3K and Akt downstream. Activated Akt regulates the activity
of numerous targets, including kinases, transcription factors and other
regulatory molecules, one of which involves the translocation of glucose
transporter-4 (GLUT4) to the membrane [48].
Fig. 9. Dose-response curve of 2-ethyl-1,4-
dimethoxybenzene (left) and reference drug
(right) inhibitory activities against 𝛼-amylase,
𝛼-glucosidase, and HMG-CoA reductase. The
reference drug for 𝛼-amylase and 𝛼-glucosidase
assays was acarbose, whereas simvastatin was
used for the HMG-CoA reductase inhibitory as-
say. Data are presented as mean ± standard de-
viation of three independent experiments.
E.I. Ugwor, A.S. James, A.I. Amuzat et al.
These network-based analyses provide insights into the targets and
mechanisms potentially involved in the anti-T2DM effects of Nigerian
propolis.
3.3. Inhibitory activities of 2-ethyl-1,4-dimethoxybenzene against key
T2DM-associated targets
Furthermore, to validate the anti-T2DM potential of Nige-
rian propolis, we evaluated the inhibitory activity of 2-ethyl-1,4-
dimethoxybenzene against 𝛼-amylase, 𝛼-glucosidase, and HMG-CoA re-
ductase, compared it to referenced drugs (Fig. 9). HMGR and 𝛼-amylase
are among the 167 overlapping targets associated with NP anti-T2DM
activity; acarbose and simvastatin, FDA-approved drugs for manag-
ing T2DM, are inhibitors of 𝛼-glucosidase and HMGR, respectively.
2-ethyl-1,4-dimethoxybenzene showed high inhibitory activity against
𝛼-amylase and 𝛼-glucosidase, with IC50 values lower than acarbose.
Although the IC50 value of simvastatin (reference drug for HMGR
assay) was lower, 2-ethyl-1,4-dimethoxybenzene showed appreciable
HMGR inhibitory activity (IC50 = 111.2 ± 1.76 μg/ml compared to
60.54 ± 3.22 μg/ml for simvastatin). These results are concordant with
our network analyses and previous reports of propolis from Nigeria
[10,14, 15] and other regions such as China [9], Taiwan [11], Morocco
[49], and Brazil [50].
3.4. Limitations of the study
A significant limitation of our study is that we did not evaluate the
effects of the identified bioactive compounds on cell viability nor their
effect at the gene/protein level. However, our study provides a pipeline
of propolis compounds that be appraised for their anti-diabetic poten-
tials via in vivo and clinical studies.
4. Conclusion
The network pharmacology approach, which holistically consid-
ers the complex interactions in biological systems, was employed to
elucidate the bioactive constituents of Nigerian propolis and provide
insights into the mechanism of their anti-T2DM activity. We iden-
tified 44 compounds (out of 202) that interacted with anti-T2DM
targets, with 2-Ethyl-1,4-dimethoxybenzene, borneol, 3,8-dihydroxy-
9‑methoxy-pterocarpan, kaempferol, decanoic acid, methyl ester,
resveratrol, 13-tetradece-11-yn-1-ol, broussonin B, 1,5,9,9-tetramethyl-
1,4,7-cycloundecatriene, and 2-(7-Octenyl)oxirane as the top ten com-
pounds. On the other hand, HSP90AA1, JUN, ESR1, STAT3, CYP3A4,
PTGS2, RELA, VEGFA, CYP2C9, and PPARG were identified as the core
anti-T2DM targets (n = 167) associated with Nigerian propolis con-
stituents. The targets were primarily involved in pathways pertinent to
lipid metabolism, regulation, and signal transduction, while 49.3% of
the targets were localised in the plasma membrane. Thus, we provide
thorough mechanistic insights into the potential anti- T2DM activities of
the Nigerian propolis’ constituents while revealing a pipeline of bioac-
tive compounds that can be developed for the therapeusis of type 2 di-
abetes mellitus.
Funding
This research did not receive any specific grant from the public, com-
mercial, or not-for-profit funding agencies.
Supplementary File 1 Nigerian propolis compound database com-
piled from previously reported works and FDA-approved diabetic drugs
used in the present study.
Supplementary File 2 Pharmacologically relevant characters of the
96 screened compounds in Nigerian propolis.
Supplementary File 3 Comprehensive lists of targets retrieved from
the databases.
9
Pharmacological Research - Modern Chinese Medicine 5 (2022) 100183
Supplementary File 4 CytoHubba analysis of the compound-target
and protein-protein interaction networks.
Supplementary File 5 GO and KEGG pathways information ex-
tracted from the DAVID bioinformatics resources.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Emmanuel I. Ugwor: Conceptualization, Writing – original draft,
Project administration, Methodology, Formal analysis, Visualization.
Adewale S. James: Conceptualization, Project administration, Method-
ology, Writing – review & editing. Adekunle I. Amuzat: Project admin-
istration, Methodology, Resources. Emmanuel O. Ezenandu: Writing
– original draft, Methodology, Resources. Victory C. Ugbaja: Writing
– original draft, Methodology, Resources. Regina N. Ugbaja: Supervi-
sion, Writing – review & editing, Resources.
Data Availability
All data generated or analysed during this study are included in this
published article and its Supplementary Files.
Acknowledgments
None.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.prmcm.2022.100183.
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