Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183

Contents lists available at ScienceDirect

Pharmacological Research - Modern Chinese Medicine

journal homepage: www.elsevier.com

F
Network pharmacology-based elucidation of bioactive compounds in
propolis and putative underlying mechanisms against type-2 diabetes

OO
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

PR
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

ARTICLE INFO ABSTRACT

Keywords: Propolis is a common remedy in Chinese medicine. We have previously demonstrated the anti-diabetic potentials
Nigerian propolis of Nigerian propolis. However, the underlying mechanisms against type-2 diabetes mellitus (T2DM) remain un-
D
Network pharmacology clear. 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
TE

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 lo-
calised in the plasma membrane and cytoplasm and primarily involved in regulatory, signal transduction and cel-
EC

lular response 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 phar-
macological 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.
RR

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
CO
ronmental, genetic and epigenetic factors and is associated with several
metabolic diseases, such as obesity and overweight. It is also a risk fac-
tor for heart disease – the leading cause of death globally [5].
Although T2DM prognosis has improved and several anti-T2DM
therapies have been formulated over the years, its prevalence continues
to surge [6]. Pharmacological agents, such as sulfonylureas, glucagon-

2019, there were 437.9 million reported cases of T2DM, with the high-
est proportion in low- and middle-income countries [3]. T2DM is a
chronic metabolic condition brought on by impaired insulin production
by pancreatic beta cells and insulin resistance in peripheral insulin-
sensitive organs [4]. The aetiology of T2DM stems from various envi-
like peptide1 agonists, HMG-CoA reductase inhibitors, and α-
glucosidase inhibitors, and lifestyle modification have been the main-
stream anti-T2DM therapies [6]. However, these agents are limited by
adverse side effects and high costs. Besides, they often target one aspect
of the otherwise multifaceted complications associated with T2DM [7].

✰ Type of Paper: Original article.

⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil.
E-mail address: ugworei@funaab.edu.ng (E.I. Ugwor).

https://doi.org/10.1016/j.prmcm.2022.100183
2667-1425/© 20XX

Note: Low-resolution images were used to create this PDF. The original images will be used in the final composition.
E.I. Ugwor et al.
Conversely, natural products, encompassing herbal remedies, possess
few side effects and have increasingly been exploited by researchers in
developing therapeutic agents against T2DM [8].
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 anti-
cancer properties [11,12]. However, the chemical constituent of propo-
lis is highly divergent, depending heavily on the bee species, the flora
surrounding the collection site, and geographical zones [13]. In Nige-
ria, propolis has purportedly been used to treat ringworm, chickenpox,
and measles and manage diabetes [12]. We have previously demon-
strated the anti-diabetic potentials of Nigerian propolis (NP), including
hypoglycemic, hypolipidemic, and hepatoprotective properties, in rats
with streptozotocin-induced diabetes [13]. Other studies have reported
similar anti-diabetic properties of NP [10,12,15]. However, the under-
PR
lying 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
natural products, thus revealing the putative mechanisms of action
against different diseases via interacting targets and associated path-
D
ways [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 nat-
TE
ural products via a compound–target–pathway-disease network con-
structs [16,19].
Therefore, this study utilised a network-based pharmacology analy-
sis to unravel the possible mechanisms of NP anti-T2DM activity and
identify responsible bioactive components. In addition, the inhibitory
EC
activity of NP's bioactive component against some targets identified
was also evaluated.
2. Methods
2.1. Assembling of compounds database for Nigerian propolis
RR
A database of ligands in Nigerian propolis was compiled from previ-
ously reported works conducted on the characterisation of propolis
within Nigeria. The canonical SMILES of each compound were re-
trieved 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
CO
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
analyses. In addition, 37 FDA-approved drugs for T2DM were from
DrugBank (https://go.drugbank.com), and their pharmacological prop-
erties were computed with SwissADME (Supplementary File 1). Princi-
pal component analysis (PCA) was conducted on the calculated proper-
ties [17]; the two most significant principal components were used to
2
Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183
compare the chemical space between the screened compounds and
drugs in a scatterplot.
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 Target Prediction (STP;
http://www.swisstargetprediction.ch/) databases [23]. On the other
hand, using the Homo sapiens mode and “type 2 diabetes mellitus” as
F
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
OO
(KEGG; https://www.kegg.jp) databases [18]. Subsequently, overlap-
ping targets between the compound-associated and ND-associated tar-
gets were identified using VENNY 2.1 (https://bioinfogp.cnb.csic.es/
tools/venny/) and used for network construction.
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 follow-
ing 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 domi-
ciled in Cytoscape.
The overlapping targets were mapped onto the Comparative Toxi-
cogenomics 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 Discov-
ery (DAVID; https://david.ncifcrf.gov/tools.jsp) server was used for en-
richment 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), biologi-
cal process (BP), molecular function (MF), and KEGG pathways perti-
nent 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.
E.I. Ugwor et al.
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
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
products 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) con-
taining 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
PR
(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 solu-
tion (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
D
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 con-
taining 400 μM NADPH and 400 μM HMG-CoA substrate in a final vol-
TE
ume of 2 mL of 100 mM potassium phosphate buffer, pH 7.4 (contain-
ing 120 mM KCl, 1 mM EDTA, and 5 mM DTT). The rate of NADPH dis-
appearance was monitored at 340 nm for 15 min. The blanks contained
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:
EC
2.7. Data management
RR
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
obtained from the in-vitro analyses into a dose-response curve to deter-
mine IC50 values.
CO
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
Supplementary File 1. The compounds were then screened using the
ADME properties, three drug-likeness approaches, and projected oral
bioavailability. A compound's ADME features predict its disposition in-
side an organism, contributing to its pharmacological (or toxicological)
action [33]. The oral bioavailability indicates the fraction of the com-
pound that will reach systemic circulation unchanged following oral
administration, which correlates with its efficacy. Furthermore, the
rules of Lipinski, Ghose, and Verber are drug-likeness approaches used
3
Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183
to identify whether a chemical molecule with a particular pharmaco-
logical or biological activity has chemical and physical qualities, such
as molecular 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 ex-
cluded.
The chemical space occupied by these screened compounds was
then compared to those of FDA-approved drugs using the two most sig-
F
nificant principal components, which explained 53.0% and 26.0% of
variances, respectively (Fig. 1). Although both groups occupied two dis-
tinct clusters, there were clear overlaps between the two groups, further
OO
illustrating 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 tar-
gets), 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 Supple-
mentary File 3).
These common targets were then entered into the STRING database
to analyse the protein-protein interactions, which comprise direct
(physical) and indirect (functional) associations, obtained from compu-
tational prediction, inter-organismal knowledge transfer, and associa-
tions 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 eliminating isolated nodes and edges, with an average node de-
gree of 4.49. The substantial number of interactions between the targets
suggests that they may be biologically related, and the anti-T2DM ef-
fects of the compounds may involve multiple targets. The top 10 targets
(hereafter referred 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 (Supplemen-
tary 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). Besides, several studies have highlighted the impor-
tance of these targets to T2DM. Lazaro et al. [34] reported that pharma-
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.
E.I. Ugwor et al. Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183

studies have reported the roles of CYP, PTGS2, and VEGFA in diabetes
[38–40]. ESR1 mediates the effects of estrogens on glucose homeostasis
[41].
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
eicosanoids) metabolism; cluster 2 nodes are primarily involved in cel-
lular responses to stimuli, such as hypoxia (HIF-1), inflammation
(NFKB1, STAT3, and RELA), heat (HSP90), proliferation (VEGF,

F
PDGFRB, PRKCZ and PPARG), and tissue remodelling (MMPs and
VEGFA). Clusters 3 and 4 were involved in signal transduction and alco-
hol metabolism, respectively. Nodes in cluster 5 have been correlated to

OO
amyloid deposition, while those in clusters 6 and 7 regulate adenylate
cyclase/cGMP downstream 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 pro-
teins.
Interestingly, of the 96 compounds, only 44 had targets common to
T2DM-associated targets (Table 1), whereas 52 compounds had no

PR
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
D
disease-associated targets (DAT).
cological inhibition of HSP90 ameliorated diabetes-associated compli-
cations, while Yang et al. [35] revealed decreased Hsp90 expression in
TE
overlap with T2DM targets. The interactions between overlapping tar-
gets and the screened compounds were visualised in a CT network (Fig.
5A), consisting of 221 nodes (44 compounds and 167 targets) and 620
interactions, with an average of 3.52 targets/compound. The overlap of
targets reflects the multi-target characteristics of the bioactive com-
pounds. 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 com-
pound-target interactions (227 of 310).
The overlapping targets were then linked to biochemical pathways

isolated rat islets exposed to high glucose. They surmised that Hsp90
might be involved in high glucose-induced islet dysfunction. Mean- using the CTD online server; the highly significant T2DM-linked path-
while, the cross-talk between JUN, RELA, and STAT3 has been impli- ways were visualised as a network with the targets and associated path-
cated in the progression of T2DM [36,37]. Clinical and pre-clinical ways represented as nodes and the associations as edges (Fig. 6). In this
EC
RR
CO

Fig. 3. Protein-protein interaction network (A.) and the top ten (core) type-2 diabetes disease targets (B.) associated with screened compounds in Nigerian propo-
lis. Core targets were identified based on the number of degrees, betweenness, and closeness using the degree algorithm of the CytoHubba plugin within Cy-
toscape (version 3.9.1).

4
E.I. Ugwor et al. Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183

F
OO
PR
D
TE

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.

Table 1
Bioactive compounds in Nigerian propolis against type-2 diabetes mellitus, as identified by network pharmacology analysis.
Rank Compound Parameters* Rank Compound Parameters*
EC

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- 27 7772.28 79.23 25 Calycosin 2 67.46 54.95
pterocarpan
4 Kaempferol 25 5349.30 72.02 26 6-Prenylnaringenin 2 56.79 51.70
RR

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- 9 2062.24 61.59 31 Vestitol 1 0.00 53.89
Cycloundecatriene
10 2-(7-Octenyl)oxirane 9 1824.00 63.34 32 Quinine 1 0.00 46.30
CO

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
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- 1 0.00 1.00
2-yl carbonate
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).

5
E.I. Ugwor et al. Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183

F
OO
PR
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.
D
TE
EC
RR
CO

Fig. 6. Target-pathway network. Dark green nodes = pathway, blue nodes = targets.

way, the 143 targets were mapped to 43 canonical pathways, with 576
interactions between these components, further highlighting the di-
verse 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 mem-
brane, and nucleoplasm, while 14.5% can be found in the mitochon-
drion. Regulatory, 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 ampli-
fies or transmits, usually involving signalling cascades of protein inter-

6
E.I. Ugwor et al. Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183

F
OO
PR
D
TE

Fig. 7. Top 10 components of Gene Ontology (GO) enrichment analyses of Nigerian propolis compounds’ anti- type-2 diabetes disease targets. (A) Cellular compo-
nent; (B) Biological process; and (C) Molecular function.
EC

actions, 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 tran-
scription and apoptosis, while 10.8% and 12.7% were negative regula-
tors of these processes, respectively. Meanwhile, 16.9 and 13.3% were
RR

involved in G-protein coupled receptor (GPCR) signalling and signal

transduction, while 12.7% and 12.0% were associated with inflamma-
tory and drug response, respectively. These biological processes corre-
spond with MP annotation, revealing that 75.3% of the targets’ func-
tions involved protein binding, a central regulatory and signalling
mechanism. Other functions include binding to DNA, zinc, and ATP,
CO

protein homodimerisation, kinase, and GPCR activities, all of which are

involved in signalling and regulation. Indeed, T2DM develops from dys-
functional insulin signalling and has been associated with impaired reg-
ulatory 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
presented in a bubble plot against the fold enrichment (Fig. 8). 31.9%
of the targets were involved in metabolic pathways, which is unsurpris-
ing given that T2DM is a metabolic disease [43]. Differentially ex-
pressed genes linked to neuroactive ligand-receptor interaction (12.7%
of targets) have been reported in diabetic patients [44,45]. Advanced
glycation end products (AGEs) interact with their receptor (RAGE) to
potentiate crosstalk that amplifies T2DM-associated complications
Fig. 8. Bubble plot showing top 10 highly enriched pathways obtained from Ky-
oto encyclopaedia of Genes and Genomes (KEGG) pathway assessment.
NLRI = neuroactive ligand-receptor interaction.
[36]. In contrast, 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 character-
istic 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, pro-

7
E.I. Ugwor et al.
PR
D
TE
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 assay. Data
are presented as mean ± standard deviation of three independent experiments.
moting the activation of PI3K and Akt downstream. Activated Akt regu-
lates the activity of numerous targets, including kinases, transcription
EC
factors and other regulatory molecules, one of which involves the
translocation of glucose transporter-4 (GLUT4) to the membrane [48].
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
RR
T2DM-associated targets
Furthermore, to validate the anti-T2DM potential of Nigerian propo-
lis, 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
CO
are among the 167 overlapping targets associated with NP anti-T2DM
activity; acarbose and simvastatin, FDA-approved drugs for managing
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. Al-
though 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 Nige-
ria [10, 14, 15] and other regions such as China [9], Taiwan [11], Mo-
rocco [49], and Brazil [50].
8
Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183
F
OO
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 considers
the complex interactions in biological systems, was employed to eluci-
date the bioactive constituents of Nigerian propolis and provide in-
sights into the mechanism of their anti-T2DM activity. We identified 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 compounds.
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
bioactive compounds that can be developed for the therapeusis of type
2 diabetes mellitus.
E.I. Ugwor et al.
Funding
This research did not receive any specific grant from the public,
commercial, 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
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.
CRediT authorship contribution statement
Emmanuel I. Ugwor : Conceptualization, Writing – original
draft, Project administration, Methodology, Formal analysis, Vi-
PR
sualization. Adewale S. James : Conceptualization, Project ad-
ministration, Methodology, Writing – review & edit-
ing. Adekunle I. Amuzat : Project administration, Methodol-
ogy, Resources. Emmanuel O. Ezenandu : Writing – original
draft, Methodology, Resources. Victory C. Ugbaja : Writing –
original draft, Methodology, Resources. Regina N. Ugbaja : Su-
pervision, Writing – review & editing, Resources.
D
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
TE
ence the work reported in this paper.
Data Availability
All data generated or analysed during this study are included in this
EC
published article and its Supplementary Files.
Acknowledgments
None.
Supplementary materials
RR
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.prmcm.2022.100183.
References
CO
[1] A.A. Gunaid, A.M. Assabri, Prevalence of type 2 diabetes and other
cardiovascular risk factors in a semirural area in Yemen, East. Mediterr. Health J.
14 (1) (2008) 42–56. https://apps.who.int/iris/handle/10665/117407.
[2] WHO, Noncommunicable Diseases, World Health Organization, 2020. https://
www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases.
Accessed June 27 2022,.
[3] S. Safiri, N. Karamzad, J.S. Kaufman, A.W. Bell, S.A. Nejadghaderi, M.J. Sullman,
et al., Prevalence, deaths and disability-adjusted-life-years (DALYs) due to type 2
diabetes and its attributable risk factors in 204 countries and territories,
1990–2019: results from the global burden of disease study 2019, Front.
Endocrinol. 3 (2021) 838027, https://doi.org/10.3389/fendo.2022.838027.
[4] U. Galicia-Garcia, A. Benito-Vicente, S. Jebari, A. Larrea-Sebal, H. Siddiqi, K.B.
Uribe, et al., Pathophysiology of type 2 diabetes mellitus, Int. J. Mol. Sci. 21 (17)
(2020) 6275, https://doi.org/10.3390/ijms21176275.
[5] D. Himanshu, W. Ali, M. Wamique, Type 2 diabetes mellitus: pathogenesis and
genetic diagnosis, J. Diabetes Metab. Disord. 19 (2) (2020) 1959–1966, https://
doi.org/10.1007/s40200-020-00641-x.
[6] F. Ventura, C. Borghi, Diabetes in the third millennium: prognosis improves, but
juvenile forms are more frequent, Eur. Heart J. Suppl. 21 (Suppl B) (2019), B17
https://doi.org/10.1093/eurheartj/suz006, J Eur Soc Cardiol.
[7] A.S. Wickramasinghe, P. Kalansuriya, A.P. Attanayake, Nanoformulation of
9
Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183
plant-based natural products for type 2 diabetes mellitus: from formulation design
to therapeutic applications, Curr. Ther. Res. Clin. Exp. 96 (2022) 100672, https://
doi.org/10.1016/j.curtheres.2022.100672.
[8] S. Kumar, A. Mittal, D. Babu, A. Mittal, Herbal medicines for diabetes
management and its secondary complications, Curr. Diabetes Rev. 17 (4) (2021)
437–456, https://doi.org/10.2174/1573399816666201103143225.
[9] Y.Z. Shi, Y.C. Liu, Y.F. Zheng, Y.F. Chen, J.J. Si, M.L. Chen, et al., Ethanol extract
of Chinese propolis attenuates early diabetic retinopathy by protecting the
blood–retinal barrier in streptozotocin-induced diabetic rats, J. Food Sci. 84 (2)
(2019) 358–369, https://doi.org/10.1111/1750-3841.14435.
[10] M.I. Oladayo, Nigerian propolis improves blood glucose, glycated hemoglobin
A1c, very low-density lipoprotein, and high-density lipoprotein levels in rat models
of diabetes, J. Intercult. Ethnopharmacol. 5 (3) (2016) 233–238, https://doi.org/
F
10.5455/jice.20160502065029.
[11] L.H. Chen, Y.W. Chien, M.L. Chang, C.C. Hou, C.H. Chan, H.W. Tang, et al.,
Taiwanese green propolis ethanol extract delays the progression of type 2 diabetes
mellitus in rats treated with streptozotocin/high-fat diet, Nutrients 10 (4) (2018)
OO
503, https://doi.org/10.3390/nu10040503.
[12] C.S. Alaribe, T. Esposito, F. Sansone, A. Sunday, I. Pagano, A.L. Piccinelli, et al.,
Nigerian propolis: chemical composition, antioxidant activity and α-amylase and α-
glucosidase inhibition, Nat. Prod. Res. 35 (18) (2021) 3095–3099, https://doi.org/
10.1080/14786419.2019.1682576.
[13] S. Huang, C.P. Zhang, K. Wang, G.Q. Li, F.L Hu, Recent advances in the chemical
composition of propolis, Molecules 19 (12) (2014) 19610–19632, https://doi.org/
10.3390/molecules191219610.
[14] R.N. Ugbaja, T.P. Fatokun, D.I. Akinloye, A.S. James, O.O. Onabanjo, O.A.
Akinloye, Propolis ethanol extract abrogates hyperglycemia, lipotoxicity, and
lowered hepatic poly (ADP-ribose) polymerase protein level in male albino rats, J.
Diabetes Metab. Disord. 20 (1) (2021) 683–696, https://doi.org/10.1007/s40200-
021-00800-8.
[15] I.R. Babatunde, A. Abdulbasit, M.I. Oladayo, O.I. Olasile, F.R. Olamide, B.W.
Gbolahan, Hepatoprotective and pancreatoprotective properties of the ethanolic
extract of Nigerian propolis, J. Intercult. Ethnopharmacol. 4 (2) (2015) 102.
[16] R.S. Ibrahim, A.A. El-Banna, Network pharmacology-based analysis for
unraveling potential cancer-related molecular targets of Egyptian propolis
phytoconstituents accompanied with molecular docking and in vitro studies, RSC
Adv. 11 (19) (2021) 11610–11626, https://doi.org/10.1039/D1RA01390D.
[17] B. Gogoi, D. Gogoi, Y. Silla, B.B. Kakoti, B.S. Bhau, Network pharmacology-based
virtual screening of natural products from Clerodendrum species for identification
of novel anti-cancer therapeutics, Mol. Biosyst. 13 (2) (2017) 406–416, https://
doi.org/10.1039/C6MB00807K.
[18] V.M. Kutluay, N.Y. Diker, Constitution of a comprehensive phytochemical profile
and network pharmacology based investigation to decipher molecular mechanisms
of Teucrium polium L. in the treatment of type 2 diabetes mellitus, PeerJ 8 (2020)
e10111, https://doi.org/10.7717/peerj.10111.
[19] M. Liao, H. Shang, Y. Li, T. Li, M. Wang, Y. Zheng, et al., An integrated approach
to uncover quality marker underlying the effects of Alisma orientale on lipid
metabolism, using chemical analysis and network pharmacology, Phytomedicine
45 (2018) 93–104, https://doi.org/10.1016/j.phymed.2018.04.006.
[20] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug. Deliv. Rev. 23 (1–3) (1997) 3–25,
https://doi.org/10.1016/S0169-409X(96)00423-1.
[21] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple,
Molecular properties that influence the oral bioavailability of drug candidates, J.
Med. Chem. 45 (12) (2002) 2615–2623, https://doi.org/10.1021/jm020017n.
[22] A.K. Ghose, T. Herbertz, J.M. Salvino, J.P. Mallamo, Knowledge-based
chemoinformatic approaches to drug discovery, Drug Discov. Today 11 (23–24)
(2006) 1107–1114, https://doi.org/10.1016/j.drudis.2006.10.012.
[23] J. Wang, X. Chen, W. Bai, Z. Wang, W. Xiao, J Zhu, Study on mechanism of
Ginkgo biloba L. leaves for the treatment of neurodegenerative diseases based on
network pharmacology, Neurochem. Res. 46 (7) (2021) 1881–1894, https://
doi.org/10.1007/s11064-021-03315-z.
[24] P. Shannon, A. Markiel, O. Ozier, N.S. Baliga, J.T. Wang, D. Ramage, N. Amin, B.
Schwikowski, T. Ideker, Cytoscape: a software environment for integrated models
of biomolecular interaction networks, Genome Res. 13 (11) (2003) 2498–2504,
https://doi.org/10.1101/gr.1239303.
[25] D. Szklarczyk, A.L. Gable, K.C. Nastou, D. Lyon, R. Kirsch, S. Pyysalo, et al., The
STRING database in 2021: customizable protein-protein networks, and functional
characterization of user-uploaded gene/measurement sets, Nucleic Acids Res. 49
(D1) (2021) D605–D612, https://doi.org/10.1093/nar/gkaa1074.
[26] P. Bernfeld, Enzymes of starch degradation and synthesis, Adv. Enzymol. Relat.
Subj. Biochem. 12 (1951) 379–428, https://doi.org/10.1002/
9780470122570.ch7.
[27] E. Apostolidis, Y.I. Kwon, K. Shetty, Inhibitory potential of herb, fruit, and
fungal-enriched cheese against key enzymes linked to type 2 diabetes and
hypertension, Innov. Food Sci. Emerg. Technol. 8 (1) (2007) 46–54, https://
doi.org/10.1016/j.ifset.2006.06.001.
[28] D. Iqbal, M.S. Khan, A. Khan, M. Khan, S. Ahmad, A.K. Srivastava, et al., In vitro
screening for β-hydroxy-β-methylglutaryl-coa reductase inhibitory and antioxidant
activity of sequentially extracted fractions of Ficus palmata forsk, Biomed. Res. Int.
(2014) 762620, https://doi.org/10.1155/2014/762620.
[29] B. Lawal, O.K. Shittu, A.N. Abubakar, I.A. Olalekan, A.M. Jimoh, A.K.
Abdulazeez, Drug leads agents from methanol extract of Nigerian bee (Apis
mellifera) propolis, J. Intercult. Ethnopharmacol. 5 (1) (2016) 43–48, https://
doi.org/10.5455/jice.20151208122127.
E.I. Ugwor et al.
[30] R.M. Omar, J. Igoli, A.I. Gray, G.U. Ebiloma, C. Clements, J. Fearnley, et al.,
Chemical characterisation of Nigerian red propolis and its biological activity
against Trypanosoma brucei, Phytochem. Anal. 27 (2) (2016) 107–115, https://
doi.org/10.1002/pca.2605.
[31] I.A. Duru, Comparative phytochemical analysis of brown, green and red propolis
from Umudike, Abia State Nigeria, Adv. J. Chem. Sect. B 3 (1) (2020) 86–97,
https://doi.org/10.22034/ajcb.2021.121910.
[32] O.S. Balogun, A.O. Oluduro, Z. Liu, Chemical composition and in vitro biological
studies of volatile oils from Nigerian bee propolis, J. Apic. Res. (2020) 1–9, https://
doi.org/10.1080/00218839.2020.1862392.
[33] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci. Rep. 7 (1) (2017) 1–3, https://doi.org/10.1038/srep42717.
[34] I. Lazaro, A. Oguiza, C. Recio, B. Mallavia, J. Madrigal-Matute, J. Blanco, et al.,
Targeting HSP90 ameliorates nephropathy and atherosclerosis through suppression
of NF-κB and STAT signaling pathways in diabetic mice, Diabetes 64 (10) (2015)
3600–3613, https://doi.org/10.2337/db14-1926.
[35] X. Yang, Y. Zhang, W. Xu, R. Deng, Y. Liu, F. Li, et al., Potential role of Hsp90 in
rat islet function under the condition of high glucose, Acta Diabetol. 53 (4) (2016)
621–628, https://doi.org/10.1007/s00592-016-0852-2.
[36] N.M. El-Nasr, D.O. Saleh, S.S. Mahmoud, S.M. Nofal, R.M. Abdelsalam, M.M.
Safar, et al., Olmesartan attenuates type 2 diabetes-associated liver injury: cross-
talk of AGE/RAGE/JNK, STAT3/SCOS3 and RAS signaling pathways, Eur. J.
Pharmacol. 874 (2020) 173010, https://doi.org/10.1016/j.ejphar.2020.173010.
[37] A. Kowalczuk, N. Bourebaba, K. Kornicka-Garbowska, E. Turlej, K. Marycz, L.
Bourebaba, Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and
PR
hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/
JNK pathway, Cell Commun. Signal. 19 (1) (2021) 1–26, https://doi.org/10.1186/
s12964-021-00735-w.
[38] S. Gravel, J.L. Chiasson, J. Turgeon, A. Grangeon, V. Michaud, Modulation of
CYP 450 activities in patients with type 2 diabetes, Clin. Pharmacol. Ther. 106 (6)
(2019) 1280–1289, https://doi.org/10.1002/cpt.1496.
[39] N. Sellami, L.B. Lamine, A. Turki, S. Sarray, M. Jailani, A.K. Al-Ansari, M.
Ghorbel, T. Mahjoub, W.Y. Almawi, Association of VEGFA variants with altered
VEGF secretion and type 2 diabetes: a case-control study, Cytokine 106 (2018)
29–34, https://doi.org/10.1016/j.cyto.2018.03.003.
D
[40] E.M. Vázquez, N. Cobo-Vuilleumier, R.A. Legido, S. Marín-Cañas, E. Nola, A.
Dorronsoro, et al., NR5A2/LRH-1 regulates the PTGS2-PGE2-PTGER1 pathway
contributing to pancreatic islet survival and function, iScience 25 (5) (2022)
TE
EC
RR
CO
10
Pharmacological Research - Modern Chinese Medicine xxx (xxxx) 100183
104345, https://doi.org/10.1016/j.isci.2022.104345.
[41] K.C. Gregorio, C.P. Laurindo, U.F. Machado, Estrogen and glycemic homeostasis:
the fundamental role of nuclear estrogen receptors ESR1/ESR2 in glucose
transporter GLUT4 regulation, Cells 10 (1) (2021) 99, https://doi.org/10.3390/
cells10010099.
[42] S. Balakrishnan, S. Dhavamani, Prahalathan C. β-Cell specific transcription
factors in the context of diabetes mellitus and β-cell regeneration, Mech. Dev. 163
(2020) 103634, https://doi.org/10.1016/j.mod.2020.103634.
[43] T.M. Batista, N. Haider, C.R. Kahn, Defining the underlying defect in insulin
action in type 2 diabetes, Diabetologia 4 (5) (2021) 994–1006, https://doi.org/
10.1007/s00125-021-05415-5.
[44] S. Kaviarasan, S. Muniandy, R. Qvist, I.S. Ismail, Gene expression profile in
leucocytes of type 2 diabetic subjects, Biomed. Res. 20 (3) (2009) 158–165 https://
F
doi.org/10.4103/0970-938X.54834 (Aligarh).
[45] W. Qu, C. Han, M. Li, J. Zhang, L. Li, Revealing the underlying mechanism of
diabetic nephropathy viewed by microarray analysis, Exp. Clin. Endocrinol.
Diabetes 123 (6) (2015) 353–359, https://doi.org/10.1055/s-0035-1548849.
OO
[46] D.S. Carvalho, A.A. de Almeida, A.F. Borges, D.V. Campos, Treatments for
diabetes mellitus type II: new perspectives regarding the possible role of calcium
and cAMP interaction, Eur. J. Pharmacol. 830 (2018) 9–16, https://doi.org/
10.1016/j.ejphar.2018.04.002.
[47] S. Huang, J. Wang, L. Zhang, S. Tian, Y. Wang, X. Shao, et al., Ras guanine
nucleotide-releasing protein-4 promotes renal inflammatory injury in type 2
diabetes mellitus, Metabolism 131 (2022) 155177, https://doi.org/10.1016/
j.metabol.2022.155177.
[48] S.A. Khorami, A.R. Movahedi, K. Huzwah, A.M. Sokhini, PI3K/AKT pathway in
modulating glucose homeostasis and its alteration in diabetes, Biomed. Sci.
Instrum. 1 (2) (2015) 46–55.
[49] N. El Menyiy, N. Al-Wali, A. El Ghouizi, S. El-Guendouz, K. Salom, B Lyoussi,
Potential therapeutic effect of Moroccan propolis in hyperglycemia, dyslipidemia,
and hepatorenal dysfunction in diabetic rats, Iran. J. Basic Med. Sci. 22 (11) (2019)
1331 10.22038%2Fijbms.2019.33549.8004.
[50] T. Fukuda, M. Fukui, M. Tanaka, T. Senmaru, H. Iwase, M. Yamazaki, et al.,
Effect of Brazilian green propolis in patients with type 2 diabetes: a double-blind
randomized placebo-controlled study, Biomed. Rep. 3 (3) (2015) 355–360, https://
doi.org/10.3892/br.2015.436.