Topical application of Mentha piperita essential oil accelerates wound healing in infected mice model

Abstract

This study was conducted to evaluate the effects of the prepared ointments from Mentha piperita essential oil (M. piperita) on wound healing in the infected mice models. Each circular full-thickness wound was inoculated with 25 × 10⁷ units of Staphylococcus aureus and Pseudomonas aeruginosa bacteria strains. The tissue bacterial count, histological analyses and expression levels of IL-10, TNF-α, TGF-β1, IL-1β, CCL2, CXCL1, VEGF and FGF-2 were assessed to identify the different doses of M. piperita on wound healing. Total tissue bacterial count, edema and inflammation level were declined, but the migration of fibroblasts, collagen synthesis and re-epithelization were increased in treated animals with M. piperita. The expression levels of CCL2, CXCL1, IL-1β, TGF-β1 and IL-10 genes were up-regulated in the M. piperita-treated animals compared to the control group. While the expression of TNF-α, VEGF and FGF-2 was down-regulated in comparison to the control group. This study indicated that M. piperita can be used for treatment of the infected wound.

Full text

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Inflammopharmacology
Experimental and Therapeutic Studies
ISSN 0925-4692
Inflammopharmacol
DOI 10.1007/s10787-018-0510-0
Topical application of Mentha piperita
essential oil accelerates wound healing in
infected mice model
Mohammad Modarresi, Mohammad-
Reza Farahpour & Behzad Baradaran

1 23
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Vol.:(0123456789)
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Inflammopharmacology
https://doi.org/10.1007/s10787-018-0510-0
ORIGINAL ARTICLE
Topical application ofMentha piperita essential oil accelerates wound
healing ininfected mice model
MohammadModarresi1· Mohammad‑RezaFarahpour2· BehzadBaradaran3
Received: 18 March 2018 / Accepted: 1 June 2018
© Springer International Publishing AG, part of Springer Nature 2018
Abstract
This study was conducted to evaluate the effects of the prepared ointments from Mentha piperita essential oil (M. piperita)
on wound healing in the infected mice models. Each circular full-thickness wound was inoculated with 25 × 107 units of
Staphylococcus aureus and Pseudomonas aeruginosa bacteria strains. The tissue bacterial count, histological analyses and
expression levels of IL-10, TNF-α, TGF-β1, IL-1β, CCL2, CXCL1, VEGF and FGF-2 were assessed to identify the different
doses of M. piperita on wound healing. Total tissue bacterial count, edema and inflammation level were declined, but the
migration of fibroblasts, collagen synthesis and re-epithelization were increased in treated animals with M. piperita. The
expression levels of CCL2, CXCL1, IL-1β, TGF-β1 and IL-10 genes were up-regulated in the M. piperita-treated animals
compared to the control group. While the expression of TNF-α, VEGF and FGF-2 was down-regulated in comparison to the
control group. This study indicated that M. piperita can be used for treatment of the infected wound.
Keywords Mentha piperita· Infectious wound· Pro-inflammatory cytokine· Collagen synthesis· Re-epithelization
Introduction
Each injury on the body can be notified as a wound, which
can interrupt the body structure. Wound healing is a com-
plex process, which occurs after damage to soft tissue and
may last for a long time (Godwin and Rosenthal 2014).
Different mechanisms and physiological processes modu-
late wound healing (Lauer etal. 2000). Wound healing is a
complicated process that contains various interdependent
stages, including hemostasis, inflammation, proliferation
and remodeling (Farahpour etal. 2015, 2017). Inflamma-
tory chemokines are produced by the different tissues and
leukocytes that are infiltrated in response to bacterial tox-
ins (Satish 2015). Inflammatory phase occurs following
activation of inflammatory chemokines. The TNF-α as an
inflammatory cytokine plays a major role in the first stage
of inflammatory phase. The IL-1β is a potent inflammatory
agent, which aggregates the neutrophils into the site of infec-
tion (Eo etal. 2016). Neutrophils and macrophages infiltrate
transforming growth factor beta 1 (TGF-β1), which activates
fibroblasts and starts the proliferative phase (Kimmel etal.
2010). The inflammatory chemokines are known to have
the regulator effects on leukocytes recruitment toward the
region of inflammation or infection. CXC chemokines first
aggregate neutrophils, lymphocytes, and manage the early
phases of wound healing (Charo and Ransohoff 2006). There
is a balance between pro- and anti-inflammatory cytokines
for wound healing, but the imbalance between both increases
inflammatory response and induces wounds that cannot be
healed (Satish 2015).
Growth factors are known to have an effect on the end-
ing phase of healing. Vascular endothelial growth factor
(VEGF) provokes cell migration, proliferation, and synthe-
sis of extracellular matrix proteins (Schultz and Wysocki
2009). Fibroblast growth factor-2 (FGF-2) promotes angio-
genesis in the proliferative phase of wound healing (Oryan
and Moshiri 2011).
Common antimicrobial agents such as silver sulfadia-
zine have been used to alleviate the risk of infection during
Inflammopharmacology
* Mohammad-Reza Farahpour
mrf78s@gmail.com
1 Department ofBasic Sciences, Faculty ofVeterinary
Medicine, Urmia Branch, Islamic Azad University, Urmia,
Iran
2 Department ofClinical Sciences, Faculty ofVeterinary
Medicine, Urmia Branch, Islamic Azad University,
Urmia57159-44867, Iran
3 Immunology Research Center, Tabriz University ofMedical
Sciences, Tabriz, Iran
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M.Modarresi et al.
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wound healing, but they have major limitations such as
microbial resistance. The use of medicinal plants and their
derivations are an appropriate strategy instead of common
chemical agents. Peppermint, the so-called Mentha piperita,
is known to have some pharmaceutical properties. Pepper-
mint essential oil (PEO) contains many compounds, includ-
ing menthol, menthon, isomenthol, limonene, cineol, men-
thyl acetate, beta-caryophyllene, menthofuran, terpinene,
caron, pinene, Sabinene, β-pinene, tannin, etc. (Herro and
Jacob 2010). PEO has antimicrobial, antiviral, and antifun-
gal activities against various types of bacteria and yeasts
(Liang etal. 2012). PEO showed anti-inflammatory activ-
ity in the croton oil-induced mouse ear edema model due
to its inhibitory effects on production of nitric oxide and
prostaglandin E2 (Sun etal. 2014). We hypothesized that
topical administration of PEO shortens pro-inflammatory
phase and accelerates the wound healing process. Thus, this
study aimed to evaluate the effects of topical administration
of ointment containing PEO (M. piperita) on the infected
wounds by evaluating mRNA expression profile of the genes
involved in the inflammation, collagen synthesis, as well as
remodeling and regeneration of epithelial tissue after tissue
injury.
Materials andmethods
Experimental animal
Ninety male BALB/c mice, aged 12–14weeks old and
weighting 27 ± 3g, were used. To alleviate the stress effects,
animals were transferred to experimental environment before
beginning of experiment. The mice received standard feed
and water. All the used procedures were approved by Ethi-
cal committee of Islamic Azad University, Urmia Branch
(No. IAUU 1102). The date of the animal ethic approval is
Jun 30 2016.
Preparation thePEO
The PEO was purchased from Barij Essence Company,
Kashan–Iran with Voucher specimen (No. 111). Based on
the data sheet of the company, menthol (39.80%), mentone
(19.55%), neomenthol (8.82%), menthyl acetate (8.64%),
1,8-cineole (5.81%), Trans-beta caryophyllene (2.76%),
germacrene-d (2.73%), limonene (1.12%), and beta-pinene
(0.92%) were the main compounds in PEO.
Induction ofinfected wounds
The infection excision wound model was induced as pre-
viously described by our previous study (Farahpour etal.
2017). Following common procedures of surgery, a circular
wound with diameter of 7mm was surgically created on the
dorsal surfaces of the mice. Then, an aliquot of 25 × 107 S.
aureus (ATCC 25923) and P. aeruginosa (ATCC 27853)
suspended in 50μL PBS were inoculated on the wound.
The animals were divided into five groups (per group 18
animals) as follows; (1) negative control: administrated
only yellow soft paraffin; (2) positive control: administrated
mupirocin ointment; (3), (4) and (5) administrated 2, 4 and
8% M. piperita (w/w), respectively. Also, 0.5g of each oint-
ment was topically administrated once per day, 24h after
colonization of the bacteria. Animals were subdivided into
three subgroups (n = 6) to evaluate the bacterial count and
molecular analyses in the wounded tissue.
Rate ofwound healing
The ratio of wound contraction was performed based on
our previous study (Farahpour etal. 2017). Wound closure
percentage was evaluated and final area drawn on glass was
calculated on days 4, 8, 12 and 16 as follows;
Percentage of wound closure = [(wound area on day
0 − wound area on day x)/wound area on day 0] × 100.
Histological analyses
The wound was divided into two halves on days 3, 7 and
14 after induction of wound. The samples underwent a
routine tissue passage process using ascending degrees of
alcohols and then they were finally embedded in paraffin
(Farahpour etal. 2015, 2017). Wounds with size of 5μm
thick were mounted on glass slides, dewaxed, rehydrated in
distilled water, and stained with Masson’s Trichrome. All
the histological evaluations were done by the two blinded
pathologists. The 4-scale system was semi-quantitatively
implemented to assess the changes in fibroblast prolifera-
tion, collagen formation, angiogenesis and epithelialization.
The results were finally presented in a 4-point scale as fol-
lows: 0, none; 0.5, few; 1, moderate; 2, many and 3, con-
siderable. Same methods were used to estimate the edema
ration. Finally, the edema was graded as negative (−), mild
(+), mild to moderate (++), moderate (+++), and intensive
(++++) as described in our previous work (Farahpour etal.
2017).
RNA extraction andquantitative real‑time PCR
After induction of wound, a specimen was prepared to evalu-
ate the genes expression profile on days 3 and 7. 3–5 grams
of wound tissues were placed in tubes containing RNase
solution (Qiagen, Germany). Follwing that, the samples
were promptely sent to the lab. After the homogenization,
RNA was extracted by Trizol method (Roche, Germany) as
recommended by manufacturer’s instructions. The cDNA
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Topical application ofMentha piperita essential oil accelerates wound healing ininfected…
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was synthesized from the RNA of cells by the Exiqon cDNA
Synthesis Kit according to the manufacturer’s instructions.
Samples were immediately incubated at 25°C for 5min fol-
lowed by 42°C for 60min; the reaction was finally termi-
nated by heating at 70°C for 5min. Light Cycler 96 Roche
device was used to evaluate the mRNA expression of the tar-
get genes, including IL-10, IL-1β, CCL2, CXCL1, TNF-α,
FGF-2, and VEGF. The primers sequences were IL-10, for-
ward (5′-CCA TCA TGC CTG GCT CAG CAC-3′) and reverse
(5′-TGT ACT GGC CCC TGC TGA TCC-3′); IL-1β, forward
(5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-
AGC TGC TTC AGA CAC TTG CAC-3′); CCL2, forward (5′-
ATG CAG GTC CCT GTC ATG CTT-3′) and reverse (5′-GGG
CGT TAA CTG CAT CTG GCT-3′); CXCL1, forward (5′-CAG
ACT CCA GCC ACA CTC CAA -3′) and reverse (5′-CAG CGC
AGC TCA TTG GCG ATA-3′); TNF-α, forward (5′-GAA GCT
CCC TCA GCG AGG ACA-3′) and reverse (5′- TTG GGC CAG
TGA GTG AAA GGG-3′); TGF-β1, forward (5′-CTG AAC
CAA GGA GAC GGA AT-3′) and reverse (5′-GGT TCA TGT
CAT GGA TGG TG-3′); FGF-2, forward (5′-GGA ACC CGG
CGG GAC ACG GAC-3′) and reverse (5′- CCG CTG TGG
CAG CTC TTG GGG-3′); VEGF, forward (5′-GCT CCG TAG
TA G CCG T GG TCT-3′) and reverse (5′-GGA ACC CGG CGG
GAC ACG GAC-3′).
Statistical analysis
One-way ANOVA and Duncan’s test were used to compare
the quantitative data of gene expression among different
groups. Kruskal–Wallis non-parametric test was used to
compare the severity of each pathological observation on
the sampling dates among different groups. The difference
among the groups was followed by the Mann–Whitney U
test.
Results
Wound contraction
The wound area significantly declined in the animals treated
with M. piperita and mupirocin compared to the nega-
tive control group on days 4, 8, 12 and 16 post-wounding
(Fig.1). Administration of M. piperita, especially in higher
doses, significantly decreased wound area (P < 0.05).
Total bacterial count ingranulation tissue
Topical administration of M. piperita and mupirocin could
significantly diminish the rate of total bacterial count in
comparison to the negative control group post-wounding
(Fig.2) (P < 0.05).
Pathological observations
Light microscopic analysis showed that the edema score
was diminished in all treated groups compared to the
control-sham group on all days after induction of wound
(Fig.3a). Interestingly, observations showed that the rate
of edema score dramatically decreased in higher doses in
the animals treated with M. piperita. Our light microscopic
analysis revealed that the infiltration of fibroblast cells into
the wound site was significantly increased (P < 0.05) in
all treated animals based on the time and dose (Fig.3b).
Further analyses indicated that the collagen deposition and
density in the wound site were enhanced in in the animals
treated with higher doses of M. piperita in comparison
to the control-sham group (Figs.3c, 4). The achieved
results also showed that re-epithelization was significantly
increased (P < 0.05) in animals treated with higher doses
of M. piperita in comparison to the other groups (Figs.3d,
5) on days 7 and 14 after wounding.
Fig. 1 Effect of M. piperita on circular excision wound area (mm2)
on various days of healing. n = 6 animals in each group. Data are pre-
sented as the mean ± SD. a–eSignificant (P < 0.05) differences between
marked groups on the same day
Fig. 2 Effect of M. piperita on total bacterial count on various days
of healing time. n = 6 animals in each group. Data are presented as
the mean ± SD. a–eSignificant (P < 0.05) differences between marked
groups on the same day
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M.Modarresi et al.
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mRNA expression level ofthetarget genes
The data for real-time PCR showed that CCL2 and CXCL1
mRNA expression in M. piperita-treated groups were
significantly (P < 0.05) higher, especially at dose of 8%,
in comparison to negative control group on day 7 after
wounding (Fig.6a, b). Our findings showed a significant
(P < 0.05) reduction in the mRNA expression of the TNF-α
gene in M. piperita-treated groups, especially at dose of
4 and 8%, in comparison to negative control group on day
7 after wound induction (Fig.6c). Effects of M. piperita
on genes expression in wounds showed that a significant
(P < 0.05) increase in positive control, 4% and 8% M.
piperita-treated groups in comparison with the control and
2% M. piperita-treated group, on day 7 after wound induc-
tion (Fig.6d–f). A significant (P < 0.05) downregulation
was also observed for VEGF and FGF-2 in the positive
control group and M. piperita group in comparison to con-
trol group on day 7 after induction of wound (Fig.6g, h).
Fig. 3 Effect of M. piperita on
edema (a), fibroblast infiltra-
tion (b), collagen regeneration
(c), and epithelization (d) on
the different days. a–eSignificant
(P < 0.05) differences between
marked groups on the same day
Fig. 4 Cross-section from the wound area on the eighth day after
wound creation. a1–a2 negative control, b1–b2 positive control
(mupirocin), c1–c2 2% M. piperita-treated, d1–d2 4% M. piperita-
treated, and e1–e2 8% M. piperita-treated groups. The marked area
with squares is presented in higher magnification. The cross-sections
represent intensive immune cells infiltration in the negative con-
trol and positive control groups in comparison with all doses of M.
piperita-treated groups. Increased collagen synthesis in all doses of
M. piperita-treated groups (marked in squares) (Masson’s trichrome
staining)
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Discussion
Our findings demonstrated that the migration rate of fibro-
blasts, collagen production and secretion, regeneration
of epithelial tissue, and downregulation of inflammatory
cytokines were increased in the groups treated with M.
piperita. PEO is known to have remarkable antibacterial
effects because of high levels of menthol, carvone thymol,
carvacrol and mentone. Laboratory studies have reported
that PEO inhibited the growth of standard strains of Escheri-
chia coli, S. aureus (Çetin etal. 2016), P. aeruginosa, Mic-
rococcus flavus and Shigella, Salmonella enteritidis (Jianu
etal. 2013; Shalayel etal. 2017). The innate immune sys-
tem modulates in the defence mechanism against infectious
agents, which infiltrates the immune cells such as neutro-
phils, macrophages and dendritic cells for phagocytosis
of infectious pathogens into the site of injury during the
elementary stages of the process of wound healing (Nathan
2006). The intense infiltration can be conduced by neutro-
phils in response to the presence of a large number of bacte-
ria in the wound site. Topical administration of M. piperita
is able to reduce infiltration of immune cells into the wound
region, and control inflammation of the tissue, which can be
attributed to antibacterial effects of M. piperita.
Fig. 5 Cross-section from the wound area on the 16th day after
wound creation. a Negative control, b positive control (mupirocin), c
2% M. piperita-treated, d 4% M. piperita-treated, and e 8% M. piper-
ita-treated groups. The cross-sections represent the mature epidermis,
showing papillae (arrows) and well-formed dermis with intensive
collagen synthesis in the all M. piperita-treated groups. Masson’s tri-
chrome staining. E epidermis, MD mature dermis
Fig. 6 Relative CCL2, CXCL1, TNF-α, IL-1β, IL-10, TGF-β1,
VEGF and FGF-2 mRNA expressions were measured by qRT-PCR
using the 2_ΔΔCt method and b-actin as an internal control. Illustra-
tion of the mRNA expression show that, CCL2 and CXCL1 mRNA
expression in M. piperita-treated groups were significantly (P<0.05)
higher, especially at dose of 8%, in comparison to negative con-
trol group on day 7 after wounding (a, b). Reduction in the mRNA
expression of the TNF-α gene in M. piperita-treated groups, espe-
cially at dose of 4 and 8%, in comparison to negative control group
on day 7 after wound induction (c). IL-1β, IL-10 and TGF-β1 mRNA
expression showed that a significant (P< 0.05) increase in positive
control, 4% and 8% M. piperita-treated groups in comparison with
the control and 2% M. piperita-treated group, on day 7 after wound
induction (d–f). A significant (P <0.05) downregulation was also
observed for VEGF and FGF-2 in the positive control group and M.
piperita group in comparison to control group on day 7 after induc-
tion of wound (g, h). All data is presented in the mean ± SD. The
non-italicized letters above each column indicate a difference in level
(P<0.05) per day
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M.Modarresi et al.
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Infection is a vital factor in the enhancment of inflam-
mation of the tissues and expression levels of IL-1, IL-6,
IL-17 and TNF-α (Campos and Calixto 2000). The obtained
results showed that topical administration of M. piperita,
especially in higher doses could decrease the expression of
TNF-α, but conversely raise the expression of TGF-β1 and
IL-10. The increments of TNF-α and IL-1β levels prolong
inflammatory stage of inflammatory phase during wound
healing process (Hozzeina etal. 2015). The increased lev-
els of IL-10 enhance activity of macrophages in the wound
site and accelerates wound healing process (Gillitzer and
Goebeler 2001; Sato etal. 1999). Also, TGF-β1 activates
the fibroblasts and begins the proliferative phase (Kimmel
etal. 2010). TGF-β1 not only participates in angiogenesis,
but also stimulates granulation tissue formation (Okuda etal.
1998) and re-epithelialization (Schmid etal. 1993). It seems
that M. piperita, especially in higher levels is able to acceler-
ate wound healing process by rising the levels of TGF-β1
and IL-10, and reducing the level of TNF-α.
On the other hand, CXCL1 gene encodes a surface pro-
tein in the cells as receptor (Milatovic etal. 2003). The
higher expression of CXCL1 increases CXCR receptors on
surface, which is associated with releasing growth factors
and inflammatory inhibitors, rising epithelial cells prolif-
eration, decrement of creatinine degradation, and improve-
ment of stability and integrity of the extracellular matrix
in the affected area. Our findings showed an increment in
mRNA expression of CXCL1 in the animals treated with M.
piperita. The overexpression of CCL2 in the samples treated
with M. piperita is also indicated compared to the control
group. The overexpression of CCL2 is also related to the
increment of macrophages recruitment into the site of dam-
age, which reduces the infection expansion, and ultimately
prevents inflammation/necrosis processes.
This action can be explained by monocyte chemoattract-
ant protein-1 (MCP1) as a chemokine pro-inflammatory
agent that is involved in the healing of various wounds. Pre-
vious studies indicated that treatment of wound area with
MCP1 protein leads to improvement in the response of mac-
rophages as previously reported (Fang etal. 2010; Lucas
etal. 2010; Raman etal. 2011). The reduction of the loaded
microbial into the would and alleviation of tissue inflamma-
tion are two initial factors for beginning the second stage of
wound healing process (Bielefeld etal. 2013). This phase is
responsible for the incement of vascular and renal functions,
migration of fibroblasts to the wound site, collagen produc-
tion, and movement of the epithelial cells from the edge to
the wound site (Farahpour etal. 2017). The results revealed
that topical administration of M. piperita, especially in
higher doses could increase the number of fibroblasts in the
wound region, collagen regeneration, and epithelialization
process on days 7 and 14. However, molecular analyses indi-
cated the downregulation of the angiogenesis-related gene
(VEGF), and fibroblast-recruiting gene (FGF-2) expression
in M. piperita-treated groups in comparison to the negative
control group. The mechanism of this association is unclear
yet and needs to be studied further.
Conclusion
It can be stated that topical administration of M. piperita
in high doses could reduce the inflammatory phase and
promote wound healing process in the infected wounds
using bacteria strains of S. aureus and P. aeruginosa. Topi-
cal administration of M. piperita, especially in high doses,
could provide acceptable efficiency in both pathological and
molecular phases. It is suggested that using 4% of M. piper-
ita can be considered as an economical level for preparation
of the commercial ointments.
Acknowledgement This study was extracted from DVM thesis of Mr
Mohammad Moddaresi in Veterinary Faculty of Islamic Azad Univer-
sity, Urmia Branch. The authors are grateful to Dr. Farahnaz Tahery for
ointment formulation and Dr. Sheryl Thomas for the native language
edition.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of
interest.
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