Wound Medicine 18 (2017) 8–20

Contents lists available at ScienceDirect

Wound Medicine
journal homepage: www.elsevier.com/locate/wndm

Review article

Honey and epithelial to mesenchymal transition in wound healing: An

evidence-based review
Abid Nordina , Nur Qisya Afifah Veronica Sainika , Mohamed S. Zulfarinab ,
Isa Naina-Mohamedb , Aminuddin Saimc , Ruszymah Bt Hj Idrusa,*
a
Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia (The National University of Malaysia), Cheras, Kuala Lumpur, 56000,
Malaysia
b
Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia (The National University of Malaysia), Cheras, Kuala Lumpur, 56000,
Malaysia
c
Ear, Nose & Throat Consultant Clinic, Ampang Puteri Specialist Hospital, Ampang, Selangor, 68000, Malaysia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 5 May 2017 This review inspects all the available report on association of honey and the epithelial mesenchymal
Received in revised form 20 June 2017 transition (EMT) event in wound healing. A systematic review of the literature was conducted to identify
Accepted 21 June 2017 relevant studies on honey and EMT. The search was done in Medline via Ebscohost and Scopus database
Available online 23 June 2017 to obtained relevant articles published between 1823 and 2017. The main inclusion criteria were research
articles published in English that reported changes in the EMT markers in in vitro wound healing model
Keywords: when subjected to honey. The literature search identified 19 potentially relevant articles, whereby 8 met
Honey the inclusion criteria. Two types of in vitro wound healing model were included, cutaneous and corneal
Epithelial
wound. All studies reported a positive effect of honey in wound healing. In terms of EMT markers, honey
Mesenchymal
was shown to upregulate mesenchymal markers and downregulated epithelial markers providing the
Wound healing
evidence of EMT activation by honey. However, the effects on EMT varied dependent on the floral origin of
the honey. This review provides good evidence that honey regulates the process of EMT and has a positive
impact on wound healing.
© 2017 Elsevier GmbH. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1. Wounds that do not heal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2. Types of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3. Features of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4. Markers of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.5. Molecular mechanism of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.6. Honey and its constituent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.7. Honey and wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1. Literature review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2. Selection of research articles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3. Inclusion and exclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4. Data extraction and management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3. Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1. Search results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2. Study characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4. Honey and EMT of cutaneous wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

* Corresponding author.
E-mail address: ruszyidrus@gmail.com (R. Bt Hj Idrus).

http://dx.doi.org/10.1016/j.wndm.2017.06.003
2213-9095/© 2017 Elsevier GmbH. All rights reserved.
 A. Nordin et al. / Wound Medicine 18 (2017) 8–20 9

5. Honey in tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6. Honey in corneal tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7. Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1. Introduction widely accepted as a hallmark of the EMT [7]. Following loss of cell
to cell adhesion, cell motility is marked by the synthesis of various
1.1. Wounds that do not heal cytoskeletal proteins and rearrangement of actin fibres. Vimentin
is the intermediate filament protein that is expressed in various
In 1986, Dr. Harold F. Dvorak first described cancer as wound cells including fibroblasts [4].
that do not heal after reporting histological similarities between Other markers of EMT that has been summarised by Michael
the two events [1]. Post Dvorak, more report on the similar cellular Zeisberg and Eric G. Neilson in their 2009 review includes the
and biochemical processes associated with both wound and to increase expression of mesenchymal/fibroblast markers such as
those off cancer development emerged [2]. One of the notable fibroblast-specific protein-1 (FSP1), discoidin domain receptor
characteristics of cancer cell is the ability to transition between tyrosine kinase 2 (DDR2), heat shock protein-47 (HSP47), collagen
immotile epithelial state to motile mesenchymal state, which I, or collagen II, or the loss of epithelial markers such as CArG box-
enables the cancer cell to invade the surrounding tissue during binding factor-A (CBF-A), lymphoid enhancer-binding factor (LEF),
metastasis [2]. This process which is known as epithelial- ß-catenin, cytokeratin, zona occludens 1 (ZO-1), and the increase
mesenchymal transition (EMT) is also an integral component in expression of transcriptional factors such as Snail, Slug, or Twist
the wound healing process [2]. [7].
Activation of EMT then triggers the release of chemokines and
1.2. Types of EMT matrix metalloproteinases (MMPs), notably MMP-2, MMP-3, and
MMP-9 by the activated cells [9]. Eventually, this will lead to
Recent report suggested that the cells within certain epithelia basement membrane damage and focal degradation of collagen IV
during specific steps of embryogenesis and organ development and laminin in the surrounding microenvironment which enables
appear to be plastic and thus able to move back and forth between cell migration [9]. Inhibition of the regulator of the MMP, the tissue
epithelial and mesenchymal states via the processes of EMT and inhibitor of metalloproteinase (TIMP) is also a paramount feature
mesenchymal to epithelial transition (MET) [3]. In light of the of EMT [7].
reported occurrence of the EMT process in these three distinct
biological events, a proposal to classify EMTs into three different 1.5. Molecular mechanism of EMT
subtypes was discussed at a 2007 meeting on EMT in Poland and a
subsequent meeting in March 2008 at Cold Spring Harbor Associations of two signalling pathways, namely Smad-depen-
Laboratories [4]. EMT process involved in embryogenesis and dent pathway and mitogen-activated protein kinase (MAPK)-
organ development, the one that do not involved fibrosis nor dependent pathway in the activation of EMT have been reported
induces an invasive phenotype resulting in systemic spread via the [10,11]. Inhibition of TGF-ß receptor, a potent inducer of EMT,
circulation was classified as type 1 EMT [4]. Type 2 EMT was resulted in the suppression of activin-like kinase (ALK) type 2, 3,
designated for EMT related to wound healing and tissue and 6, as well as the Smad-4 and -5 suggesting their involvement in
regeneration. This is the EMT event in response to trauma and EMT activation [12]. Involvement of p38 MAPK and RhoA were
inflammatory injuries [4]. The main characteristic of type 3 EMT reported in autocrine TGF-ß–induced EMT in NMuMG mouse
related to cancer progression is the occurrence of genetic and mammary epithelial cells. Integrin beta 1 signalling is necessary
epigenetic changes, particularly the ones favouring clonal out- for TGF-ß activation of p38MAPK and in mouse epithelial cell
growth and the development of localised tumours [4]. plasticity [13]. The connection between inflammation and EMT
was demonstrated when cyclooxygenase-2 (COX-2) was shown to
1.3. Features of EMT inactivate Smad signalling and enhance EMT stimulated by TGF-ß
through a prostaglandin E2 (PGE2)-dependent mechanism [14].
Primary features of EMT include loss of cell to cell adhesion, loss
of planar and apical-basal polarity, as well as an increase in cell 1.6. Honey and its constituent
mobility and migration capacity [2]. In type 2 EMT, polar epithelial
cells turn into motile fibroblast under the induction of EMT ligand Honey is a highly viscous, supersaturated sugar solution
such as tumour growth factor-ß (TGF-ß) [5]. Removing EMT- derived from a mixture of various nectar of flowers or plant
inducing ligand restored epithelial phenotype of the cells secretion by the honeybee, Apis mellifera. It is made up of
suggesting reversibility of EMT, which is later known as MET [6]. approximately 80% carbohydrate and 20% water [15]. About 180
different substance including amino acids, vitamins and minerals
1.4. Markers of EMT have been reported in honey [16]. The composition of honey varies
sensibly dependent on its floral origin. In the past, researchers used
Defining specific markers of EMT has been the subject of flavonoids content to determine the floral origin of honey [17,18].
interest in recent years to ensure similar phenomena was Variation in the honey flavonoid has been reported within honey of
evaluated among all EMT researchers [7]. E-cadherin is an similar floral origin [17]. This variation was influenced by the
important feature of epithelial cells enabling it to form cell to extraction and detection method used [19].
cell adhesion [8]. Loss of the E-cadherin expression and increase Among all honey, the monofloral Manuka honey has been the
expression of N-cadherin, known as cadherin switch has been subject of interest within the international scientific community in
10 A. Nordin et al. / Wound Medicine 18 (2017) 8–20
recent years [19]. In terms of flavonoid compound, several studies
reported detection of pinobanksin, chrysin and pinocembrin that
made up the majority of the total phenolic compound in Manuka
honey. Luteolin, kaempferol, quercetin, 8-methoxykaempferol,
isorhamnetin and galangin were also detected in minute amount
[20–22]. The findings agree with earlier study made using honey
collected from different geographical location throughout the
world [18].
1.7. Honey and wound healing
In terms of wound healing, the clinical efficacy of honey is well
established [23–26]. The beneficial benefit of honey in wound
healing is believed to be due to its anti-inflammatory, antioxidant,
antibacterial and tissue growth stimulating properties which have
been extensively studied in the past decade [27]. It is believed that
the benefit of honey in wound healing is due to the favourable
microenvironment it created to the wound bed [27]. Typically,
honey has a pH ranging from 3 to 4. This acidic condition is not only
unfavourable for bacterial growth but also shown to stimulate
macrophage bactericidal action, reduce protease activity, increase
fibroblast activity, and increase oxygenation [27]. Honey high
osmolality in combination with its high sugar content has also
been shown to stun bacterial growth, spawning the extensive
research that focuses on the antibacterial effect of honey [28–30].
In spite of a vast amount of literature reporting clinical efficacy
of honey in wound healing, the underlying mechanism of its action
are still largely obscure. Exploring direct stimulation of honey on a
particular cellular mechanism such as EMT helps in understanding
its effect on cellular function. In this review, studies reporting
direct stimulation of EMT by various types of honey and its
constituent will be discussed.
2. Methods
2.1. Literature review
A systematic review of the literature was conducted to identify
relevant studies about the reported effect of honey on the
epithelial to mesenchymal transition event within cells, particu-
larly in wound healing. To conduct a comprehensive search of
biomedical science journals, we used Medline via Ebscohost
(published between 1865 and January 2017) and Scopus (published
between 1823 and January 2017) databases. The search strategy
involved a combination of the following two sets of keywords [1]
honey* OR pinocembrin* OR pinobanksin* OR chrysin* OR
methylglyoxal* AND [2] epithelial* OR mesenchymal* OR cad-
herin* OR vimentin* OR metalloproteinase*.
2.2. Selection of research articles
The results were limited to studies that were published in
English language that included abstracts. Review article, news, case
report was excluded from the review. For this review, only studies
that reported [1] the association of honey and the epithelial to
mesenchymal transition in an [2] in vitro model of wound healing
that is [3] measuring changes in markers of the EMT were included.
2.3. Inclusion and exclusion criteria
For the purpose of this review, only studies that reported direct
effect of honey on markers of EMT is included. Occurrence of EMT
is marked by the down regulation of epithelial markers and up
regulation of mesenchymal marker. Studies included must
measure the changes of at least one of the epithelial or
mesenchymal markers which are (1) cadherin switch, (2)
cytoskeletal protein vimentin expression, or (3) activity or
expression of the metalloproteinase. EMT can occur in multiple
events in the body such as cancer metastasis, organ fibrosis, and
embryonic development. In terms of wound healing, there are
many types of model available whether in vitro or in vivo. The
presence of confounding factors in in vivo model might obscure the
role of honey in EMT. For the purpose of this review, studies that
employ in vivo model that reports the effect of honey on the EMT
events of (1) cancer metastasis, (2) organ fibrosis, or (3) embryonic
development were excluded from this study.
2.4. Data extraction and management
Papers were screened in three phases before included in the
review. In the first phase, any paper that did not match the
inclusion criteria based solely on the title was excluded. In the
second phase, abstracts of the remaining papers were screened and
papers that did not meet inclusion criteria were excluded. In the
final phase, the remaining papers were read thoroughly by four
independent reviewers to exclude any paper that did not meet
inclusion criteria. All reviewers must agree on the inclusion of
selected articles for the review before the data extraction phase
begins. Any differences in opinions were resolved through
discussion between the reviewers. In order to standardise the
data collection, all data extraction was performed independently
with the use of a data extraction form. The following data were
recorded from the studies: [1] type of honey use; [2] type of cells
used; [3] a brief description of the methods used in the study; [4] a
brief description of the results of the study; [5] conclusion of the
study.
3. Result
3.1. Search results
The literature searches identified 1316 potentially relevant
articles. Four reviewers independently assessed all articles for
inclusion or exclusion based on title and abstract. 1297 of these
articles were excluded because they were not related to honey or
not related to EMT in wound healing. From the remaining 19
articles, 9 duplicate articles were removed before the full papers
were retrieved for thorough reading. Two articles were identified
as the outdated version of the current study by Abd Ghafar 2016
[31,32] and Barui 2014 [33,34]. Differences of opinion between the
reviewers regarding the inclusion or exclusion of the full articles
were resolved by discussion. A total of 8 articles were retrieved for
further assessment and data extraction to be included in this
review. A flow chart of the selection process, including reasons for
exclusion, is shown in Fig. 1.
3.2. Study characteristics
The summary of the characteristics of all studies is displayed in
Table 1. All studies were published between the year 2010 and
2016. Two types of wound healing were modelled among the
studies included, cutaneous and corneal wound. Six studies focus
on cutaneous wound with five studies used immortalised human
keratinocyte HaCat cells [33,35–38] and one study used primary
keratinocyte derived from human penis foreskin [39]. Two studies
focused on corneal wound with corneal cells derived from New
Zealand white rabbit [31,40].
In terms of honey used, the type of honey used varies from one
study to another. In terms of country of origin, three studies
 A. Nordin et al. / Wound Medicine 18 (2017) 8–20 11

Search of electronic databases:

MEDLINE via Ebscohost, SCOPUS

Identification of abstracts:
MEDLINE via Ebscohost = 576 SCOPUS= 740 Total = 1 316

Exclusion of studies that is not related to honey or its bioactive

compound or not related to epithelial to mesenchymal transition of
wound healing: 1 297

Selected abstracts:
MEDLINE via Ebscohost = 9 SCOPUS= 10 Total = 19

Removal of duplicates: 9

Full text articles obtained:

MEDLINE via Ebscohost = 9 SCOPUS = 1 Total = 10

Fulfill inclusion criteria

Full text articles read thoroughly:

MEDLINE via Ebscohost = 9 SCOPUS = 1 Total = 10

Removal of two studies which were outdated versions of Abd

Ghafar 2016 and Barui 2014.

Full text articles included in the review:

MEDLINE via Ebscohost = 7 SCOPUS = 1 Total = 8

Fig. 1. Flowchart of the selection pro.

obtained their honey from India [33,35,36], two from Malaysia
[31,40], two from Slovakia [38,39], and one from Japan [37]. The
study that obtained their honey from Japan compared the
difference of three distinct honey in their study [37]. In terms of
floral origin, three studies used Acacia honey in their study
[31,37,39]. Other honey floral origin includes Buckwheat [37],
Manuka [37], Gelam [40], Jamun [35], and Fir honeydew [38]. Two
studies from India, Barui 2014 and Chaudhary 2015 did not
mention the floral origin of the honey they used [33,36].
4. Honey and EMT of cutaneous wound healing
Ranzato et al. is the first to report direct modulation of honey in
the EMT process of HaCat cells wound healing [37]. In their study,
three types of honey were compared namely acacia, buckwheat
and manuka. In terms of re-epithelialisation, similar improvement
was observed among different types of honey. In terms of cell
signalling mechanism employed by honey, the use of kinase and
calcium inhibitors revealed the onset of varying cellular mecha-
nisms by each honey. This variation among different honeys was
also observed in terms of the phosphorylated protein activation,
MMP as well as EMT gene expression suggesting the complexity of
recommending one honey over another. All honey samples used by
Ranzato et al. was from Japan.
One of the important phase of cutaneous wound healing is the
inflammation phase. An earlier study by Majtan et al. measured the
effect of acacia honey on the expression of the cytokines tumour
necrosis factor-a (TNF-a), interleukin-1 ß (IL-1ß), and TGF-ß as
well as MMP-9 in human keratinocyte [39]. In this study, acacia
honey promoted the upregulation of all measured cytokines
including TGF-ß and MMP-9. More recent study by Majtan et al.
later revealed decreased MMP-9 secretion when HaCat cell lines
was treated with fir honeydew honey [38]. This seemingly
contrasting findings might suggest that honey can regulate the
inflammation process. Honey used in both studies originated from
Slovakia.
Two studies by Chaudhary et al. has been included in this
review [35,36]. Both studies use HaCat cells as an in vitro model of
cutaneous wound healing. In one study, Jamun honey was
supplemented to HaCat cells under hypoxic or normoxic condition.
Supplementation of honey resulted in greater wound healing in
both conditions with higher wound closure rate observed in 0.1%
honey dilution under hypoxia as compared to normoxia [35].
Upregulation of p63 and E-cadherin and lesser expression of
hypoxia-inducible factor 1a (HIF-1a) was also observed in both
normoxic and hypoxic condition of honey treatment compared to
the control [35]. In another study, six honey samples were
characterised for its physicochemical properties according to the
Codex Alimentarius [36]. Honey collected from Himalaya Drug
Company was selected and increased wound closure rate was
observed with its supplementation in HaCat cells. This effect was
accompanied by upregulated expression of p63, E-cadherin, and
b-catenin with predominantly cytoplasmic expression of its
protein. E-cadherin upregulation was corroborative with
12 A. Nordin et al. / Wound Medicine 18 (2017) 8–20

Table 1
Summary of the study included.

No Articles Type of honey Type of cells Methodology Results Conclusion

1 Abd Ghafar Acacia honey purchased from
2016 Ministry of Agriculture, Malaysia.
Honey were diluted in culture
medium.
Rabbit Treatment groups:
stromal
derived
corneal
fibroblasts.
1) Basal medium (FD).
2) Basal medium with serum
(FDS).
3) Basal medium with 0.025 %
acacia honey (FD + AH).
4) Basal medium with both
serum and 0.025 % acacia honey up to day 6.
(FDS + AH).
Parameters:
1) Wound closure rate was
measured via scratch assay.
2) Gene expression of ALDH,
vimentin, a–SMA, collagen I,
lumican and MMP12 were
measured using reverse
transcriptase polymerase chain
reaction (RT–PCR).
3) Protein expression of ALDH, e) Increasing lumican gene
vimentin, and a–SMA were
evaluated using
immunocytochemistry.
1) Supplementation of 0.025 % Honey accelerates corneal
acacia honey improved fibroblast migration and
percentage of wound reduction differentiation in corneal
by wound healing model.
a)7.9 % and 8.7 % in FD at day 3
and 6 respectively
b) 5.4 % and 18.5 % in FDS at day
3 and 6 respectively.
2) Supplementation of 0.025 %
acacia honey alters gene
expression during wound
healing by
a) Decreasing ALDH expression
b) Increasing vimentin
expression up to day 6.
c) Increasing a-SMA expression
at day 3 that is later subsided at
day 6.
d) Increasing collagen I
expression up to day 6.
expression up to day 6.
f) Increasing MMP12 expression
at day 3 that is later on subsided
at day 6.
3) Immunocytochemistry
analysis revealed that
supplementation of 0.025 %
acacia honey alters protein
expression during wound
healing by
a) Decreasing expression of
ALDH up to day 6.
b) Increasing expression of
vimentin up to day 6.
c) Increasing expression of
a-SMA up to day 6.

2 Yusof 2016 Gelam honey from the Apiary
Unit, Department of Agriculture,
Malaysia. Honey were diluted in
culture medium.
Rabbit Treatment groups:
stromal
derived
corneal
keratocytes.
1) Basal medium (BM).
2) Basal medium with serum
(BMS).
3) Basal medium with 0.0015 %
gelam honey (BM + GH).
4) Basal medium with both
serum and 0.0015 % gelam
honey (BMS + GH).
Parameters:
1) Cell proliferation was
measured via 3-(4,5–
dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
(MTT) assay.
1) Supplementation of gelam Honey induces the
honey at the concentration transition of keratocyte into
between 0.0004 % and 0.0031 % corneal fibroblasts.
significantly increased cell
proliferation with both BM and
BMS.
2) Supplementation of 0.0015 %
gelam honey alters gene
expression during wound
healing by
a) Increasing ALDH expression.
b) Increasing vimentin
expression.
c) Decreasing a– SMA
expression.
3) Immunocytochemistry
analysis revealed that
supplementation of 0.0015 %
gelam honey alters protein
expression during wound
healing by
a) Decreasing expression of
ALDH.

Table 1 (Continued)
No Articles Type of honey
3 Chaudhary Monofloral jamun honey
2015 collected from Societe naturelle, keratinocyte
India. Honey were diluted into
culture medium.
4 Chaudhary Honey collected from the
2015 Himalaya Drug Company, India.
Honey was diluted in culture
medium.
A. Nordin et al. / Wound Medicine 18 (2017) 8–20
Type of cells Methodology
2) Genes expression of ALDH,
vimentin, and a–SMA was
measured using RT–PCR.
3) Proteins expression of ALDH,
vimentin and a–SMA was
measured using
immunocytochemistry.
HaCat human Treatment groups:
cell lines.
1) Culture medium.
2) 1 % honey.
3) 0.1 % honey.
4) 0.01 % honey.
5) All dilution under hypoxia
condition.
Parameters:
1) Antioxidant properties of the 5) Supplementation of honey
honey dilution will be estimated caused
using nitro blue tetrazolium
chloride (NBT) reduction assay.
2) Keratinocyte survival rate
was measured via MTT assay.
3) Cell cycle analysis was
measured using flow–
cytometry.
4) Wound closure rate was
measured via scratch assay.
5) Protein expression of p63, E- 6) Supplementation of honey
cadherin, and HIF–1a was
measured via
immunocytochemistry.
6) Gene expression of p63, E-
cadherin, and HIF–1a was
measured using RT-PCR.
HaCat human Treatment groups:
keratinocyte
cell lines.
1) Culture medium.
2) 0.01 % honey.
3) 0.004 % honey.
4) 0.1 % honey.
5) 0.25 % honey.
Parameters:
1) Honey physico–chemical
characterization was done with
various assay following the
13
Results Conclusion
b) Increasing expression of
vimentin.
c) Decreasing expression of a–
SMA.
1) Maximum antioxidant Honey supplementation
activity was observed at honey was important in hypoxic
concentration of 0.01% and 0.1 % wound healing.
in both normoxia and hypoxia.
2) HaCat cell can survive at
maximum of 0.1 % honey under
normoxic condition.
3) Supplementation of 0.1 %
dilution of honey induced the
cell cycle toward more G2/M
(proliferative) phase in
normoxia and up to some extent
in hypoxia.
4) Supplementation of honey
increased wound closure rate
a) Highest at 0.1 % dilution.
b) Lowest at 1 % dilution.
c) In both hypoxia and normoxia
but to a greater extent in
hypoxia.
a) Translocalization of E-
cadherin from membranous to
cytoplasmic at 0.1 and 0.01 %
dilution in both normoxic and
hypoxic condition.
b) Increased nuclear expression
of p63 protein at 0.1 % dilution
in both normoxic and hypoxic
condition.
c) Decreased nuclear expression
of HIF-1a in both 0.1 and 0.01 %
dilution in normoxic condition.
In hypoxic condition, 0.1 %
dilution showing more robust
decrease compared to the
control.
influence gene expression by
a) Increasing E-cadherin
expression.
b) Increasing p63 expression.
c) Decreasing HIF–1a
expression.
1) Among the six samples Physico–chemical
collected, sample A from properties of honey as well
Himalaya Drug Company was as their appropriate
selected. Sample A showed dilutions are important in
providing the most
optimum effect in wound
healing.
a) acidic pH (3.9).
b) conductivity (8.03 mS/cm ).
c) ash content (0.890 mg).
d) viscosity (230500 cP).
e) moisture content (12.48 w/
w).
f) proteins (2.82 %).
g) carbohydrates (76.40 %).
14 A. Nordin et al. / Wound Medicine 18 (2017) 8–20
Table 1 (Continued)
No Articles Type of honey Type of cells Methodology
suggestion from Codex
Alimentarius.
2) Cell viability was measured
via MTT assay.
3) Wound closure rate was
measured via scratch assay.
4) Protein expression of E–
cadherin, ß–catenin, and p63
was measured via
immunocytochemistry.
5) Gene expression of p63, E-
cadherin, ß-catenin, GnT–III,
and GnT–V was measured via
RT–PCR.
5 Barui 2014 Honey spun into fiber together HaCat human Treatment groups:
with alginate. Origin not stated. keratinocyte
and 3T3
human
dermal
fibroblast cell
lines.
1) Honey -alginate (1:1) fiber
(HAF).
2) Alginate fiber (AF)
3) Plain surface.
Parameters:
1) Cell viability for both HaCat
and 3T3 cells were measured via
MTT assay.
2) Expression of E–cadherin,
collagen-I and collagen-III were
evaluated in both HaCat and 3T3
cells via immunocytochemistry.
6 Majtan Fir honeydew honey from Medar Treatment groups:
2013 apiary, Bardejov, Slovakia
Results Conclusion
h) phenols (240 mg GAE/100g).
i) flavonoids (1.27 mg QE/100g).
j) highest free radical
scavenging activity (72.10 % in
300 ml).
2) Cell survival rate was
improved at 0.01 %, 0.04 %, 0.1 % ,
and 0.25 % honey concentration.
Maximum viability was
observed at 0.1 % concentration.
3) Overall wound closure was
a) Highest in 0.1 % honey
concentration compared to
control.
b) Lowest in 0.25 % honey
concentration compared to
control.
4) Immunocytochemistry
analysis revealed
a) Upregulation of nuclear
expression of p63 in 0.1 % honey
but suppressed in 0.25 % honey.
b) Upregulation of cytoplasmic
but not membranous
expression of E–cadherin was
observed in 0.01 % and 0.1 %
honey but suppressed in 0.25 %
honey.
c) ß-catenin membranous
expression showed a gradual
decrease from 0.01 % to 0.25 %
honey. Cytoplasmic expression
of ß–catenin followed similar
pattern as E–cadherin.
5) Gene expression analysis
revealed
a) Upregulation of p63.
b) Upregulation of E–cadherin.
c) Upregulation of b–catenin.
d) Downregulation of GnT–III.
e) Upregulation of GnT–V.
1) MTT assays revealed the non- Honey–alginate fiber
toxicity of the scaffold with the improved wound healing at
highest viability observed in the molecular levels in both
HAF group in both cells. keratinocyte and dermal
fibroblasts.
2) Immunocytochemistry
analysis revealed
a) In HaCat, increased
expression of E–cadherin,
predominantly membranous,
when cultured on HAF and AF
compared to the cytoplasmic
expression in control.
b) In 3T3, increased expression
of collagen I and III is detected
when cultured on HAF and AF
compared to the control.
Honey treatment has the
potential to regulate
 A. Nordin et al. / Wound Medicine 18 (2017) 8–20 15

Table 1 (Continued)
No Articles Type of honey Type of cells Methodology Results Conclusion
HaCat human 1) HAE had no cytotoxic effect overproduction of MMP in
keratinocyte on the cells at concentrations up chronic wound.
cell lines to 2.5 mg/ml.
1) Honey aqueous extract (HAE) Among the phenolic
at compounds tested, treatment of
apigenin and kaempferol
yielded a similar effect to HAE in
terms of
a) 0.1 mg/ml 2) Dose-dependent reduction of
the protein expression of MMP–
9. This is accompanied by
increased expression of the
inhibitor TIMP–1.
b) 0.5 mg/ml 3) Dose-dependent reduction of
the activity of MMP–9.
c) 1 mg/ml 4) Dose-dependent reduction of
the gene expression of MMP–9.
d) 2 mg/ml
e) 2.5 mg/ml
2) Phenolic acids
a) p–coumaric acid
b) ferulic acid
3) Flavonoids
a) quercetin
b) apigenin
c) kaempferol
d) isorhamnetin
e) chrysin
f) naringenin
g) galangin
4) dimethyl sulfoxide (DMSO)
Parameters:
1) Cell viability was measured
with Alamar blue assay.
2) Activity of MMP-9 was
measured using gelatin
zymography
3) Protein expression of MMP9
was measured using Western
blot.
4) Gene expression of MMP 9
was measured using RT-PCR.

7 Ranzato Acacia, buckwheat, and manuka
2012 honey obtained from Yamada
Apiculture Centre, Japan
HaCat human Treatment groups:
keratinocyte
cell lines
1) Culture medium
2) 0.1 % acacia
3) 0.1n % buckwheat
4) 0.1 % manuka
5) All honey with kinase and
calcium inhibitors.
Parameters:
1) Cell viability was measured
using calcein–
acetoxymethylester (AM) assay. strongest inhibition with all
2) Wound closure rate was
measured via scratch assay.
Inhibitors of the ERK, p38, c-Jun
N-terminal kinase (JNK), PI3K,
1) Calcein-AM assay data Honey induces EMT in
showed similar low cytotoxicity wound repair but varies
levels for all honeys. among different types of
honey.
2) Wound closure rate
a) Significantly increase in 0.1 %
concentration of all honey
compared to the control.
b) Among different types of
honey, the wound closure rate
was similar.
c) All honey was sensitive to the
calcium inhibitor resulting in
the complete abolishment of the
wound healing effect.
d) Acacia honey at 0.1 %
concentration showed the
lowest reduction in wound
healing compared to the
control. It is the most sensitive
to the inhibitor of ERK.
e) Buckwheat honey at 0.1 %
concentration was inhibited to a
greater extent than acacia. It is
also more sensitive to the ERK
inhibitor.
f) Manuka honey at 0.1 %
concentration showed the
drugs
3) Cell migration rate revealed
16 A. Nordin et al. / Wound Medicine 18 (2017) 8–20
Table 1 (Continued)
No Articles Type of honey Type of cells Methodology
and mTOR kinases, as well as
calcium were also used to
elucidate the molecular
pathway involved in wound
healing.
3) Cell migration was measured
by counting the cells that did
not migrate via the trans-well
plate.
4) Production of syndecan–4
was measured via enzyme–
linked immunosorbent assay
(ELISA).
5) MMP enzymes expression
was evaluated using antibody
array against 7 types of MMP
and 3 types of TIMP.
6) Phosphorylated proteins
expression was evaluated using
antibody array against 95
different types of kinases.
7) EMT gene expression was
evaluated using The Human
Epithelial to Mesenchymal
Transition (EMT) RT2 ProfilerTM
PCR Array
Results Conclusion
a)Significantly increase in 0.1 %
concentration of all honey
compared to the control.
b) Among different types of
honey, 0.1 % buckwheat honey
increased the migration the
most.
4) Syndecan-4 expression was
significantly increased in 0.1 %
buckwheat honey but not the
others.
5) The MMP antibody array
revealed 0.1 % honey induced
a) Significant upregulation of
MMP–9 expression for all
honey.
b) Upregulation of MMP–3 was
the highest for manuka honey.
c) Downregulation of MMP–10
and MMP–13 was the greatest
for acacia honey.
d) Downregulation of TIMP–4
was the greatest for buckwheat
honey.
6) Phosphorylated protein
antibody array revealed 0.1 %
honey induced
a) Activation of CDK2 by all
honey.
b) Activation of FAK by all honey.
c) Activation of G3BP–1 by all
honey.
d) Activation of VASP by
buckwheat honey.
e) Activation of integrin–b3 by
manuka honey.
f) Activation of cdc25C by
buckwheat honey.
g) Activation of p42/44 by
buckwheat and acacia honey.
7) The EMT gene profiler
revealed varying expression
among honeys
a) 0.1 % acacia honey induced
upregulation of 12 genes
(WNT5, JAG1, AHNAK, FZD7,
MMP3, STEAP1, VIM, HPRT1,
TFPI2, MMP9, CDH2, PLEK2) and
downregulation of 22 genes
(FGFBP1, NOTCH1, WNT11,
ERBB3, IGFBP4, KRT19, TGFB2,
TGFB3, SPARC, VPS13A, BMP1,
CAMK2N1, F11R, TMEM132A,
KRT7, OCLN, AKT1, RGS2,
RPL13A, MMP2, PTK2, SMAD2).
b) 0.1 % buckwheat honey
induced upregulation of 2 genes
(MMP3, HPRT1) and
downregulation of 7 genes
(TGFB2, NODAL, FGFBP1, TFPI2,
SPARC, TGFB3, IGFBP4).
c) 0.1 % manuka honey induced
upregulation 13 genes (WNT11,
MMP3, MMP9, FN1, VIM, HPRT1,
MSN, ITGA5, TGFB1, TFPI2,
STEAP1, TMEM132A, MST1R)
and downregulation of 11 genes
 A. Nordin et al. / Wound Medicine 18 (2017) 8–20 17

Table 1 (Continued)
No Articles Type of honey Type of cells Methodology Results Conclusion
(WNT5B, TGFB2, FGFBP1, KRT19,
CAMK2N1, SMAD2, TGFB3,
FZD7, MITF, SPARC, RPL13A).

8 Majtan Acacia honey from Mr Peter Irha
2010 (Slovakia) and 55kDA
glycoprotein major royal jelly
protein 1 (MRJP1) of the royal
jelly obtained from Institute of
Apiculture, Liptovsky Hradok,
Slovakia.
Human skin Treatment groups:
derived
epithelial
keratinocyte
1) Culture medium
2) 1 % acacia honey
3) 5 mg/ml of MRJP1
4) 25 mg/ml MRJP1
Parameters:
1) Cytotoxic effect of honey and 3) Immunohistochemistry
MRJP1 protein was measured by analysis revealed
LDH assay.
2) Gene expression of TNF–a,
IL–1ß, TGF–ß, and MMP–9 was Reduced collagen IV detection is
evaluated using RT–PCR.
3) Protein expression of MMP-9
and collagen IV was evaluated
with immunohistochemistry.
1) Percentage of cytotoxicity is Honey has proinflammatory
less than 5 % for all dilutions of effect in skin keratinocytes.
honey and MRJP1 used.
2) Gene expression analysis
revealed
a) Both honey and MRJP1 at 25
mg/ml concentration
upregulated TNF–a expression
by 2–fold compared to the
control.
b)Honey upregulated IL–1ß by
12-fold compared to the control.
For MRJP1 treatment, the fold-
change is not significant
compared to the control.
c) Honey upregulated TGF–ß
expression by 8-fold compared
to the control. For MRJP1
treatment, the fold-change is
not significant compared to the
control.
d) Honey upregulated MMP–9
expression very significantly
compared to the control. For
MRJP1 treatment, the fold–
change is not significant
compared to the control.
a) Upregulation of MMP–9.
in accordance to the MMP–9 up-
regulation.

downregulation of N-acetylglucosaminyltransferase III (GnT-III) 6. Honey in corneal tissue regeneration

and upregulation of GnT-V [36]. All honey used by Chaudhary et al.
originated from India [35,36].
5. Honey in tissue engineering
Honey potential as a biomaterial in tissue engineering is
promising considering their regenerative prospective in the
treatment of different wound types. Barui et al. investigated the
important molecular aspects induced in keratinocytes and
fibroblast during wound healing under honey-alginate matrix
[34]. Honey-alginate matrix was fabricated via wet spinning in
calcium chloride bath. HaCat cells cultured on honey-alginate
matrix demonstrated upregulation of ki67, p63, and E-cadherin
expression through healing progression [34]. Analysis of honey
with 3T3 human dermal fibroblast cell lines revealed improved
collagen III expression with honey-alginate matrix [34]. Both of
these findings signify the improvement of wound healing with the
addition of honey in the alginate matrix and proves the benefit of
honey to be used as a scaffold in tissue engineering. Honey used by
Barui et al. originated from India [35].
Two studies have investigated the effect of honey in corneal
tissue regeneration. Abd Ghafar et al. revealed significant
acceleration of the rabbit corneal fibroblast migration and wound
healing with 0.025% acacia honey treatment [31]. This was
accompanied by the increased expression of myofibroblast
markers vimentin, collagen type I, a-smooth muscle actin
(a-SMA), and MMP-12 [31]. Conversely, alcohol dehydrogenase
(ALDH), the enzyme that ensures corneal transparency, was
reduced by acacia honey treatment [31]. The result signifies the
transition from the quiescent phenotype of corneal keratocytes
into a fibroblast repair phenotype, a characteristic of EMT within
the corneal tissue that is impedance in its wound healing. A similar
effect by 0.015% Gelam honey was also reported in corneal
keratocyte by Yusof et al. [40]. Both acacia and gelam honey used
with corneal cells originated from Malaysia [31,40].
7. Discussions
Wound healing is a complex physiological process that
includes dynamic shift between different phases namely
18 A. Nordin et al. / Wound Medicine 18 (2017) 8–20
inflammation, proliferative and remodelling. Evidence of thera-
peutic used of honey can be traced back to the ancient Egyptian
civilisation. Emergence of antibiotic resistance bacteria has pose
new challenge in clinical therapy, particularly in wound healing
intervention [41]. This leads to the need to rediscover the use of
honey in wound healing therapy. Clinical observation with honey
in broad spectrum of wound intervention has been well
established to support its use in wound treatment [23–26]. In
terms of wound healing, all studies reported a positive effect of
honey in improving wound closure rate in vitro.
With the advancement of biomedical sciences, scientists are
now able to extract more detailed information on a particular
biological process, all the way to the level of interaction between
molecules within the cells. One of the paramount biological
phenomena within the wound healing physiology is the
mobilisation of immotile epithelial cell through the process
known as EMT [2]. Understanding molecular interaction within a
biological event and how external stimuli can influence this
interaction is important in order to maximally exploit therapeutic
properties of the said external stimuli. Hence, clinical efficacy of
honey in wound healing need to be supplemented by the
molecular study of its therapeutic benefit.
In terms of association of honey and EMT, the literature search
revealed five studies that reported direct association of honey and
EMT [42–46]. However, four studies reported EMT occurrence in
cancer metastasis [42,43] and tissue fibrosis [44,45] and were
excluded from this review following our exclusion criteria. This
finding stressed the need of study focusing on the effect of honey
on Type 2 EMT of the wound healing to really understand the
effect of honey on this phenomenon.
Ranzato et al. compared the EMT modulation by honey from
three different botanical sources namely Acacia, Buckwheat, and
Manuka. Their findings shown that honey enhances wound
healing and that this honey-driven wound repair goes through
the activation of regulatory pathways of cell proliferation and
locomotion, leading to keratinocyte re-epithelialisation. All
honey was sensitive to the calcium inhibitor suggesting the
paramount role of calcium in wound healing. Manuka and
buckwheat seemed to activate p38, extracellular signal-regulated
kinase (ERK), and mechanistic target of rapamycin (mTOR)
pathways more than acacia. Phosphoinositide 3-kinase (PI3K)
pathways inhibition affect wound healing by manuka the most
but also give divergent result among all honey.
In terms of protein activation, all honeys activated cyclin-
dependent kinase 2 (CDK2), focal adhesion kinase (FAK), and
rasGAP SH3 binding protein 1 (G3BP-1) suggesting the transition
into proliferative motile phenotype triggered by all honeys.
However, activation of vasodilator-stimulated phosphoprotein
(VASP) and cdc25C by buckwheat only, integrin-b3 by manuka
honey only, as well as p42/44 mitogen activated protein kinase by
both buckwheat and acacia prove that the mode of action of
honey of different floral origin can differ. For syndecan-4, only
buckwheat honey induced significant change in the expression of
this adhesion molecule. For EMT markers, common feature
shared among all honeys include rise of hypoxanthine phosphor-
ibosyltransferase-1 (HPRT-1) and MMP-3 expressions. In general,
the EMT analysis suggested that honey can induce keratinocyte
EMT but varies sensibly among different honey and it is difficult
to recommend one honey over another.
Prior to the oldest study included in this review, study on the
effects of honey has always focused on its immunomodulatory
properties conducted using immune cells [47–49]. In 2010, Majtan
et al. was the first to investigate immunomodulation of honey and
royal jelly on human skin keratinocyte, an epithelial cell of the
epidermis [39]. They reported proinflammatory effect of honey by
measuring cytokines and MMP [39]. However, in a more recent
study using honey from different source and immortalised
keratinocyte cell lines HaCat, they observed an anti-inflammatory
effect of the honey [38]. This seemingly contradicting result might
suggest that honey can act as both pro-inflammatory and anti-
inflammatory immunomodulator depending on the type of honey.
Increased expression and activity of MMP indicated activation of
EMT [6]. Physiologically this is important to degrade surrounding
extracellular matrix (ECM) to enable cell motility. From the
findings of Majtan et al., there are biological plausibility to believe
that honey plays a role in the remodelling phase of wound healing
where the secretion and degradation of ECM is important.
Two studies by Chaudhary et al. were included in this review. In
the first study, role of oxygen supplementation was investigated in
the presence of honey [35]. Treatment of honey at 0.1%
concentration under hypoxia condition demonstrated faster
wound healing rate compared to the normal condition [35].
Interestingly, reduction of HIF-1a by honey treatment suggested a
protective effect of honey to cells subjected to hypoxia [35]. They
proposed that honey might be important in the treatment of
hypoxic wound.
In the second study, Chaudhary et al. highlighted the
importance of physicochemical characterisation of the honey
[36]. Selection of the best honey from the six samples that was
used in their study was based on Codex Alimentarius, the standard
guideline for the purity of natural products. In terms of EMT
markers, upregulation of cytoplasmic E-cadherin was observed in
both studies suggesting the transition of keratinocyte into the
motile mesenchymal phenotype [35,36]. This is accompanied by
the increase in proliferative markers p63 in both experiments
[35,36].
Tissue engineering is a discipline that refers to the practice of
combining scaffolds, cells, and biologically active molecules into
functional tissues [50]. Use of honey as a biomaterial as a scaffold
is promising considering it regenerative properties. Barui et al.
investigated the added benefit of culturing cells on honey-
alginate matrix that they fabricated [34]. Culturing 3T3 human
dermal fibroblast on honey-alginate enhanced expression of
collagen I gene, supporting the role that honey plays in ECM
remodelling [34]. For HaCat cells, honey alginate matrix induced
expression of ki67, p63, and E-cadherin gene expression indicat-
ing improved re-epithelisation [34]. In essence, Barui et al.
demonstrated that using honey as a biomaterial is proven to give
added benefit particularly in wound healing application. This
benefit of honey in wound healing application is in accordance to
a number of in vivo and clinical studies using materials fabricated
with honey [51–53].
The process of corneal healing involved the activation of
quiescent keratocytes and transition into repair phenotypes; the
corneal fibroblasts and myofibroblasts [54]. This is a feature of
EMT type 2 of the corneal wound healing [6]. Studies by Abd
Ghafar et al. and Yusof et al. using corneal keratocyte both
revealed the influence of honey on this EMT particular to corneal
regeneration [31,40]. Two honeys from two different floral origin
which is Gelam and Acacia, both from Malaysia, revealed
induction of the repair type fibroblast [31,40]. Overall, the
beneficial effects of honey on corneal regeneration might support
its use as eyedrop for injury of the eye.
8. Conclusion
The studies included in the review, all reported positive effect of
honey in wound healing. This is in accordance to its reported
clinical efficacy in wound healing. Although there are variations in
its effect depending on floral origin and the type of cells used in the
experiments, all studies included in this review indicates honey
 A. Nordin et al. / Wound Medicine 18 (2017) 8–20
has a positive impact on wound healing by driving the transition of
immotile epithelial cell into the motile mesenchymal phenotype.
Conflict of interest
The author confirms that this article content has no conflicts of
interest.
Acknowledgement
We would like to thank the Faculty of Medicine UKM for
providing resources to write this review.
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