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Research Article
Topical Administration of Manuka Oil Prevents UV-B
Irradiation-Induced Cutaneous Photoaging in Mice
Oh Sook Kwon,1Seung Hee Jung,2and Beom Seok Yang2
1Department of Integrative Medicine, Korea University Medical School, 126-1 Anam-Dong, Sungbuk-Gu, Seoul 136-705,
Republic of Korea
2Chemical Kinomics Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Sungbuk-Gu,
Seoul 136-791, Republic of Korea
Correspondence should be addressed to Beom Seok Yang; bsyang@kist.re.kr
Received February ; Revised May ; Accepted May
Academic Editor: Ki-Wan Oh
Copyright © Oh Sook Kwon et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Manuka tree is indigenous to New Zealand, and its essential oil has been used as a traditional medicine to treat wounds, fever,
and pain. Although there is a growing interest in the use of manuka oil for antiaging skin care products, little is known about its
bioactivity. Solar ultraviolet (UV) radiation is the primary environmental factor causing skin damage and consequently premature
aging. erefore, we evaluated manuka oil for its eects against photoaging in UV-B-irradiated hairless mice. Topical application of
manuka oil suppressed the UV-B-induced increase in skin thickness and wrinkle grading in a dose-dependent manner. Application
of % manuka oil reduced the average length, depth, and % area of wrinkles signicantly, and this was correlated with inhibition
of loss of collagen ber content and epidermal hyperplasia. Furthermore, we observed that manuka oil could suppress UV-B-
induced skin inammation by inhibiting the production of inammatory cytokines. Taken together, this study provides evidence
that manuka oil indeed possesses antiphotoaging activity, and this is associated with its inhibitory activity against skin inammation
induced by UV irradiation.
1. Introduction
Manuka tree (Leptospermum scoparium)isasmallshrubthat
grows in most parts of New Zealand, and its essential oil has
been used for many centuries by the native tribes and the
immigrants of New Zealand and Australia to treat wounds,
infection, inammation, fever, and pain. In vitro and in vivo
studies have suggested the oil to contain various bioactive
constituents. Among them, beta-triketons are best known to
possess high antibacterial and antifungal activities [–]. In
addition, manuka oil possesses sesquiterpene hydrocarbons
[–], which show anti-inammatory and analgesic actions
[]. Manuka oil was also known to contain substantial
amount of antioxidant compounds that can protect cell
components from the harmful action of free radicals [,,].
Currently, its commercial application for a skin care and
an antiaging product is increasing. However, scientically
controlled studies about its ecacy against skin aging are still
rare.
Solar ultraviolet (UV) radiation is one of the most harm-
ful environmental factors that cause skin damage. Repeated
exposure to UV radiation ultimately causes premature skin
aging also called photoaging, which is characterized by for-
mation of ne and coarse wrinkles, increased skin thickness,
dryness, laxity, and pigmentation [,]. e cellular and
molecular mechanisms of UV irradiation-induced photoag-
ing have been studied extensively in the last decades.
UV irradiation induces the production of reactive oxygen
species (ROS) in skin cells, which is primarily responsible
for photoaging [,]. e generated ROS activates cellular
signaling pathways to activate kinases such as p, Jun N-
terminal kinase (JNK), and mitogen-activated protein kinase
(MAPK) [,]. ese kinases ultimately stimulate the
transcriptional activities of activator protein- (AP-) and
nuclear factor- (NF-) 𝜅B[,]. Among the main target
genes of these transcriptional factors are the genes encoding
for matrix metalloproteases (MMPs) such as MMP-, MMP-
, and MMP- []. MMPs degrade collagen bers in the skin,
Evidence-Based Complementary and Alternative Medicine
and collagen bers are an important part of the connective
tissue involved in the maintenance of dermal strength and
elasticity. Reduction in collagen bers and their degradation
into fragments are important steps that lead to skin aging.
e activation of AP- has been suggested to antagonize
transforming growth factor- (TGF-) 𝛽signaling pathway
which stimulates the expressions of procollagen genes [].
UV irradiation also induces skin inammation, which in
turn leads to the release of inammatory cytokines such
as tumor necrosis factor- (TNF-) 𝛼and interleukin- (IL-)
𝛽[,]. ese cytokines can accelerate photoaging by
inducing MMP expression in addition to increasing epider-
mal hyperplasia [,]. Since the ROS generated are the
main cause of photoaging due to UV irradiation, various
antioxidant compounds have been tested to protect the skin
from photoaging. Antioxidants remove free radicals and help
repair cellular damage to an extent. Topical applications of
variousantioxidantssuchasvitaminsCandE,selenium,
soy isoavones, and polyphenolic compounds attenuated
photoaging [–].
In this study, we evaluated antiphotoaging activity of
manuka oil by using UV-irradiated hairless mice since the
oil contains antioxidant and anti-inammatory bioactive
chemicals, which we thought would prevent photoaging.
2. Material and Methods
2.1. Animals and UV-B Radiation. Six-week-old female
albino hairless mice (SKH-) were obtained from the Charles
River Laboratory (Wilmington, MA, USA) a week before
the experiment. Animals were kept at a temperature of
23 ± 1∘Cand50%±10%ofrelativehumidityinaspecic
pathogen-free environment. e mice were divided into
groups with mice in each group. e animal experiment
was approved by the institutional ethics committee for animal
care of Korea Institute of Science and Technology and was
conducted in accordance with the guidelines for the care and
use of laboratory animals (Institute of Laboratory Animal
Resources, ) as adopted and promulgated by the National
Institutes of Health. For UV-B irradiation, TLW/RS
UV lamps (Philips, Somerset, NJ, USA) with an emission
spectrum between and nm (peak: – nm) and
a Kodak cell lter to remove wavelengths of less than nm
(UVC) were used. UV-B irradiation intensity on the mouse
skin surface was measured using a UV meter (Waldmann
GmbH & Co., Villingen-Schwenningen, Germany). e irra-
diation intensity at cm from the light source was about
. mW/cm2. Initially, the minimal erythema dose (MED) to
induce erythema with sharp margins on the dorsal skin of the
mice aer was dened as MED, which was calculated to
be approximately mJ/cm2.emicewereexposedtoUV
light times per week (Monday, Wednesday, and Friday) for
a total of weeks. e radiation dose was increased weekly
by MED from MED up to MED and then maintained
at MED until the end of the experiment. Manuka oil
purchased from Coast Biologicals, New Zealand, was diluted
at dierent concentrations of %, %, and % using ethanol,
and 𝜇L of the solution was topically applied to the dorsal
area every day except Sunday for weeks. On the day of UV
irradiation, mice were treated aer UV exposure.
2.2. Evaluation of Skin ickness and Wrinkle Formation. e
dorsal skin of the hairless mice was lied up by pinching
gently, and skin thickness was measured using a caliper
(Tokyo, Japan) at weeks , , , and of the study period.
Wrinkle formation was evaluated visually by trained graders
according to the scaling grade described by Bissett et al.
atweeks,,,andduringthestudyperiod[]. e
graders were blinded to the radiation dose administered to
the mice. Before the animals were killed at the end of the -
week study period, skin surface impressions for skin wrinkle
replicas were prepared by applying silicon rubber (Silo
Dental Impression Material, Flexico Developments, Steve-
nage, Hertfordshire, UK) to the dorsal skin of the unstrained
mice. Wrinkle shadows from the impression replicas were
produced by illuminating the replica on a horizontal stand
with a light source angled at ∘,andtheimageswere
recorded and analyzed using Skin-Visiometer VL and
its soware (Courage & Khazaka, Cologne, Germany). e
average length, average depth, and the % area of wrinkles were
determined as described previously [].
2.3. Histological Analysis and Collagen Staining. e mice
were killed by cervical dislocation under anesthesia at the
end of the experiment. For histological analyses, skin spec-
imens were obtained from the central dorsum and xed
in % formalin before being embedded in paran. Five-
micrometer thick slices were sectioned from the paran-
embedded specimens and were deparanized and processed
by haematoxylin and eosin staining for histological anal-
ysis and Masson trichrome staining for the visualization
of collagen bers. Five randomly chosen elds per stained
section were photographed under a light microscope with
x magnication. By using the photographs, epidermal
thickness was measured as the distance from the basement
membrane in the interfollicular epidermis to the bottom of
the stratum corneum. Furthermore, the integrated optical
density of the collagen bers was measured using Image Pro
Plus . soware., and the % relative collagen density was
calculated by normalizing to % density value of normal
skin treated with a vehicle.
2.4. Reverse Transcriptase Polymerase Chain Reaction (RT-
PCR). Tota l R NA w a s i sol a t e d f rom sk i n b i opsy s a m p l es
stored in liquid nitrogen using Triazol (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s protocol and its
concentration was determined by measuring the absorbance
at and nm. e quality and quantity of the extracted
RNA were conrmed by electrophoresis in % denaturing
agarose gel. e oligonucleotide primer sequences for RT-
PCR analysis to estimate the murine m-RNA level of MMP-
, MMP-, and GAPDH were used as described previously
(MMP-; -ccaggtgtggggtgcctgat-and -caaacctgggcctgg-
ctgga-, MMP-; -tagcaggttatcctaaaagca-and -cca-
gctattgctcttcaat-,andGAPDH;
-cccactaacatcaaatgggg-
Evidence-Based Complementary and Alternative Medicine
and -acacattgggggtaggaaca-)[,]. For RT-PCR reac-
tions, 𝜇goftotalRNAwasincubatedwithngofrandom
hexamers, and U of reverse transcriptase (Invitrogen,
Carlsbad, CA, USA) for min at ∘C, and the reaction
was terminated at ∘C for minutes. A : dilution of the
reaction mixture containing c-DNA was subjected to PCR
amplicationusingpmolofprimersandUofamSure
Taq DNA polymerase (Gendepot, Barker, TX, USA) with the
cycling program: st cycle, min at ∘C, next cycles; s
at ∘C, s at ∘C, and s at ∘Candthenalcycle,
min at
∘C. 𝜇L of the PCR product was separated by
electrophoresis on .% agarose gel and stained with ethidium
bromide.
2.5. Preparation of the Total Protein Extract and Enzyme-
Linked Immunosorbent Assay for Cytokines. Biopsied tissues
obtainedfromthemicethatwerekilledwerestoredinliquid
nitrogen. e frozen tissues were ground into powder and
homogenized in mL of lysis buer containing mM of
Tris (pH: .), mM of NaCl, mM of EDTA, % Triton-
X-, and protease inhibitor cocktail (Roche, Indianapolis,
MN, USA). e samples were then sonicated times for s,
and the undissolved pellet was removed by centrifugation at
, g for min at ∘C. e supernatant was assayed for
TNF-𝛼and IL-𝛽by using an enzyme-linked immunosorbent
assay (ELISA) kit purchased from eBioscience (San Diego,
CA, USA). Each measurement was done in triplicate accord-
ing to the manufacturer’s instructions.
2.6. Immunohistochemical Staining of Macrophages. 𝜇m-
slices of skin tissue section were deparanized and rehy-
drated, then treated with % hydrogen peroxide solution
for min, and blocked by using % normal sera. e slides
were incubated with rabbit monoclonal antibody of CD
(Abcam, Cambridge, UK) aer : dilution, followed by
incubation with goat antirabbit antibody conjugated with
HRP ( : dilution; Gendepot, Baker, TX, USA). e
staining signals on the tissue slides were detected with DAB+
Substrate—Chromogen (DAKO, Carpinteria, CA, USA) fol-
lowing the manufacturer’s instructions. ereaer, counter-
staining was carried out with hematoxylin. Five randomly
chosen elds per each stained section were photographed
under a light microscope with x magnication and the
stained macrophages were counted, followed by calculating
the average number.
2.7. Data Analysis. Data were presented as mean ±SEM for
each treatment group and plotted using SigmaPlot soware
(Systat Soware Inc., San Jose, CA, USA). e SPSS for
Windows version . was used for the statistical analysis.
Unpaired Student’s t-test was used to compare dierences
between groups. Statistical signicance between the dier-
ent doses of manuka oil to inhibit the increase of skin thick-
ness and wrinkle grading was analyzed by using repeated-
measures analysis of variance. 𝑃 < 0.05 was considered
statistically signicant.
3. Results
3.1. Topical Application of Manuka Oil Inhibits UV-B
Irradiation-Induced Skin ickening and Wrinkle Formation
in Hairless Mice. In order to evaluate the antiphotoaging
activity of manuka oil, we topically applied 𝜇Lofmanuka
oilsolutiondilutedwithethanolin%,%,and%
concentrations which correspond to doses of . mg/kg ,
. mg/kg, and . mg/kg, respectively, or a vehicle (ethanol)
alone to UV-B-irradiated dorsal skin of hairless mice. In
the group treated with the vehicle only, the skin was rough
and scaly aer weeks of UV-B-irradiation. Furthermore,
compared to the nonirradiated control group, the group
showed thick and deep wrinkles. Moreover, there were signs
of erythema in some mice exposed to UV irradiation. In
contrast, following topical application of manuka oil, the
visible skin conditions improved in a dose-dependent man-
ner (Figure (a)). Repetitive UV irradiation was associated
with a gradual increase in skin thickening in the vehicle-
treated group, whilst the skin thickness remained almost
unchangedinthenonirradiatedcontrolgroup.However,the
increase in skin thickness was signicantly suppressed in the
group treated with manuka oil in a dose-dependent manner
(Figure (b)). We also estimated the increase in wrinkle
formation by using the visual grading method. Repetitive
exposure to UV-B radiation increased the wrinkle grade in
the mice that were treated with the vehicle alone. However,
topical treatment of manuka oil signicantly suppressed the
increase in the wrinkle grade in a dose-dependent manner
(Figure (c)). We did not observe any notable phenotypic and
behavioral adverse side eects in mice by topical treatment
with the manuka oil solutions during the experiment.
e antiwrinkle activity of % manuka oil was further
analyzed by skin replica method. Repetitive UV-B irradiation
for weeks led to the formation of deep and long wrinkles
on the skin (Figure (a)). However, topical application of
% manuka oil suppressed the wrinkle formation. When
the depth, length, and total % of wrinkles in the images
of the replicas were evaluated, signicant increases were
observed in all the parameters in the vehicle-treated group
compared to the nonirradiated control group. However,
topical treatment of % manuka oil signicantly decreased
the depth, length, and total % of wrinkles (Figures (b),(c),
and (d)).
3.2. Manuka Oil Suppresses Epidermal Hyperplasia and Pre-
vents the Loss of Fiber Collagen Content in the Skin of UV-B-
Exposed Hairless Mice. e antiphotoaging eect by manuka
oil was further evaluated by histological analysis of the
biopsied skin specimens aer staining with haematoxylin
and eosin. e stained sections obtained from the animals
treated with the vehicle alone showed a signicant increase in
epidermal thickness because of UV-B irradiation for weeks
(Figures (a) and (b)).isndingisconsistentwiththe
previously reported nding that signicant epidermal hyper-
plasia is induced during photoaging due to UV-B irradiation
[]. However, in the mice treated with % topical manuka
oil, the epidermal hyperplasia was signicantly diminished
compared to that in the group treated with the vehicle alone.
Evidence-Based Complementary and Alternative Medicine
Vehicle only 1% 5% 10%
No UV-B control
UV-B
Manuka oil
(a)
1.3
No UV-B control
0.8
0.9
1
1.1
1.2
Vehicle only
1% manuka oil
5% manuka oil
10% manuka oil
Week s
0246810
Skin thickness (mm)
0.6
0.7
(b)
No UV-B control
Vehicle only
1% manuka oil
5% manuka oil
10% manuka oil
Week s
0246810
5
Wrinkle grade
1
2
3
4
0
(c)
F : Inhibition of UV-B induced cutaneous photoaging by manuka oil in hairless mice. e dorsal skin surfaces of hairless mice were
irradiated by UV-B, times a week for weeks while various concentrations of topical manuka oil were applied every day except Sunday. Each
treated group consists of mice. (a) Representative photographs to show the appearance of the dorsal skin area at the end of weeks of the
experiment. Increase of skin thickness (b) and wrinkle grade (c) due to repetitive UV-B irradiation and their suppression by manuka oil in a
dose-dependent manner. e increases of both skin thickness and wrinkle grade were signicantly dierent between the no UV-B-irradiated
controlgroupandthegroupstreatedwith%and%ofmanukaoil(𝑃 < 0.01).
is result suggests that manuka oil suppresses epidermal
hyperplasia to attenuate photoaging.
We also evaluated the change in collagen content in the
skin tissues exposed to UV-B irradiation and the eect of
topical treatment of manuka oil on this parameter by using
Masson-trichrome staining method. UV-B irradiation for
weeks was associated with signicantly reduced density of
the stained dermal collagen bers in the tissues treated with
the vehicle compared to the normal skin tissues. However,
a signicantly higher staining density was observed with
topical administration of % manuka oil compared to vehicle
treatment alone (Figures (a) and (b)). We estimated the
expressions of MMP- and MMP- in UV-B-irradiated skin
tissues by using RT-PCR analysis. Induced elevations of
MMP- and MMP- m-RNA were observed by the chronic
UV-B irradiation. However, the inductions of both MMP-
and MMP- were notably suppressed when % manuka
oil was treated topically (Figure (c)). is result suggests
that manuka oil can suppress UV-B irradiation-induced
degradation of collagen bers and this activity is at least in
part associated with its inhibition against the UV-B-induced
elevations of metallomatrix proteases to destroy collagen
matrix. erefore, our histological data are consistent with
the results from the wrinkle analysis and conrm that
manuka oil possesses antiphotoaging activity.
3.3. Manuka Oil Inhibits the Production of Proinammatory
Cytokines and Suppresses the Inltration of Macrophages in
UV-B-Irradiated Mouse Skin. e production of the proin-
ammatory cytokines, namely, IL-𝛽and TNF-𝛼, was exam-
ined in the extract of skin biopsy tissues using ELISA.
Eight-week chronic exposure of dorsal skin of hairless mice
to UV-B increased the levels of TNF-𝛼and IL-𝛽in the
tissues treated with the vehicle alone (Figures (a) and (b)).
Evidence-Based Complementary and Alternative Medicine
UV-B
No UV-B control Vehicle only 10% manuka oil
(a)
60
65
##
40
45
50
55
35 No UV-B Vehicle Manuka oil
Mean depth (m)
*
(b)
0.6
0.7
##
Mean length (mm)
0.2
0.3
0.4
0.5
0
0.1
No UV-B Vehicle Manuka oil
**
(c)
18
Wrinkle (%)
4
6
8
10
12
14
16
##
0
2
No UV-B Vehicle Manuka oil
**
(d)
F : Analysis of wrinkles using skin replicas taken from the dorsal skin. e skin replicas of no UV-B-irradiated control (no UV-B)
group, vehicle only-treated group (vehicle), and the group that received % manuka oil (manuka oil), respectively, were generated at the end
of the experiment. (a) Representative images taken from the replicas. Analysis of the replica images was performed using the Skin-Visiometer
and its soware for the determination of mean length of wrinkles (b), mean depth of wrinkles (c), and percentage of the wrinkle area (d).
Values are me a n s ±SEM (𝑛=6per group). ##: signicantly dierent from no UV-B control group (𝑃 < 0.01). ∗and ∗∗: signicantly dierent
from vehicle only group ( ∗∗𝑃< 0.01 and ∗𝑃< 0.05).
isresultisconsistentwiththepreviousreportthatUV
irradiation induces skin inammation [,]. However, with
% topical manuka oil, induction of both cytokines was
inhibited signicantly. In addition, we investigated an extent
of macrophage inltration in the UV-B-irradiated skin by
using immunohistochemical staining. e UV-B irradiation
forweeksincreasedthenumberofmacrophagesinltrated
into the dermis (Figures (c) and (d)). However, the topical
treatment of % manuka oil inhibited the increase signi-
cantly by approximately .%. Taken together, these results
Evidence-Based Complementary and Alternative Medicine
UV-B
10% manuka oil
No UV-B control Vehicle only
(a)
80
100
##
Epidermal thickness (pixel)
0
20
40
60
No UV-B Vehicle Manuka oil
**
(b)
F : Manuka oil suppresses UV-B-induced increase in epidermal thickness. (a) Representative photographs of haematoxylin and eosin
stained dorsal skin sections taken at the end of the experiment from the no UV-B irradiation control (no UV-B) group, vehicle only group
(vehicle), and the group that received % manuka oil (manuka oil), respectively. Magnication: x fold; scale bar: 𝜇m. (b) e relative
values of epidermal thickness are estimated from the digital photoimages of haematoxylin and eosin stained sections and depicted as mean ±
SEM (𝑛=6per group). ##: signicantly dierent from no UV-B control group (𝑃 < 0.01). ∗∗: signicantly dierent from vehicle only group
(𝑃 < 0.01).
suggestthattopicaltreatmentofmanukaoilcansuppressUV-
B-inducedinammatoryreactionsintheskin.
4. Discussion
Traditionally, various medicinal activities of manuka oil have
beenwellknownandstudieshaveprovideddatatosupport
its bioactivity [–,]. In recent years, there is a growing
interest in the cosmetic properties of manuka oil, as it has
been traditionally known to maintain the elasticity of the
skin and its youthful appearance. However, there is scarcity of
scientic data to support such activities of manuka oil. In this
work,weevaluated,forthersttime,theprotectiveecacyof
manuka oil against skin photoaging induced by UV-B irradi-
ation with the help of a hairless mouse model. e degree of
the activity of dierent doses of manuka oil was quantied
by assessing skin thickness, wrinkle grade, histopathology,
and the amount of proinammatory cytokines. Our work
provides clear evidence that the topical application of manuka
oiliseectiveinattenuatingskinphotoaging.Weconrmed
that the topical treatment of % manuka oil could signi-
cantly suppress the signs of photoaging, including wrinkle
formation, epidermal thickness, and reduction in collagen
ber content.
We hypothesize the antiphotoaging activity of manuka
oilseeninourmousemodelstudytobeduetobioactivities
of chemical compounds present in the oil. Firstly, manuka
oil contains antioxidant chemicals such as 𝛾-terpinene and
terpinen--ol [,]. ese antioxidants are expected to
scavenge and directly remove ROS and therefore attenuate
the main cause of photoaging induced by chronic UV
irradiation. Skin is continuously exposed to environmental
insults since it is the rst organ to protect the body from
environment. UV from sunlight generates reactive oxygen
species (ROS) in the skin. ROS in turn induce oxidative
stress and skin inammation giving rise to various symptoms
of photoaging. For instance, wrinkle formation, a hallmark
of skin aging, occurs because of accumulated skin damages
such as matrix destruction and skin inammation. erefore,
theremovalofROSisamajortargettoprotectskinfrom
solar UV irradiation. Accordingly, the topical application
Evidence-Based Complementary and Alternative Medicine
Vehicle only
UV-B
10% manuka oilNo UV-B control
(a)
100
120
Collagen density (%)
20
40
60
80 ##
0No UV-B Vehicle Manuka oil
**
(b)
UV-B
MMP-1
MMP-3
GAPDH
No UV-B Vehicle Manuka oil
(c)
F : Manuka oil inhibits UV-B-induced reduction of collagen ber content and expression of MMPs. (a) Representative images of
collagen bers (blue color) stained by Masson-trichrome staining protocol in the skin biopsy obtained at the end of the experiment from
thenoUV-Birradiationcontrolgroup(noUV-B),vehicleonlygroup(vehicle),andthegroupthatreceived%manukaoil(manukaoil),
respectively. Magnication: x fold; scale bar: 𝜇m. (b) Relative % collagen density from each image was estimated by using Image Pro
Plus . soware. and normalizing to the density of normal skin control (%). e values are mean ±SEM (𝑛=6per each group). ##:
signicantly dierent from noUV-B control group (𝑃 < 0.01). ∗∗: signicantly dierent from vehicle only group (𝑃 < 0.01). (c) m-RNA levels
of MMP- and MMP- in the skin tissue for each group were estimated using RT-PCR of total RNA isolated from the pooled biopsies of each
group obtained at the end of the experiment. Et-Br stained bands are shown aerphotopicture. RT-PCR of GAPDH m-RNA was used for an
internal control.
of antioxidants is an approach to ameliorate UV-induced
photoaging. In fact, various botanical extracts possessing
antioxidant activity were useful in the prevention of UV-
B-mediated skin damage [,]. Epigallocatechin gallate
(EGCG), an antioxidant included in green tea, prevented
degradation of collagen bers and DNA damage induced by
UV radiation [].
Cellular signaling pathways involved in ROS induced
photoaging have been studiedin detail. ROS generated by
UV-B activates ERK/, p MAPK, and JNK signaling in
epidermal keratinocyte and dermal broblast cells [–].
ese signaling pathways eventually enhance the transcrip-
tional activities of AP- and NF-𝜅B[,,]. e activated
transcriptional factors enhance the expressions of MMPs,
which degrade skin structure by destroying collagen matrix.
erefore, blocking the signaling molecules activated due to
the generation of ROS could be an additional strategy to pre-
vent photoaging. Manuka oil contains sesquiterpene hydro-
carbons as one of the major components [–]. Interestingly,
there have been reports that some sesquiterpene compounds
can block the signaling pathways involved in oxidative stress
[,]. In this work, we showed that the topical treatment of
manuka oil inhibited the reduction of ber collagen density
caused by chronic UV-B irradiation, and this was correlated
with the suppression of the induced MMP- and MMP-
expressions. is result suggests that the reduction of MMPs
might be responsible for the prevention of loss of collagen in
thetissuetreatedwithmanukaoil.Weproposethatbioactive
chemicals included in manuka oil such as sesquiterpenes
could act to block the cell signalings generated by ROS, thus
inhibiting the activation of AP- and NF-𝜅B which are mainly
responsible for the induction of MMPs. It will be interesting
to examine if the sesquiterpenes in manuka oil could inhibit
the downstream signaling molecules modulated by ROS.
Our study suggests that manuka oil can suppress skin
inammation reactions in the UV-B-irradiated hairless
mouse model. Multiple studies have suggested UV irradiation
to induce skin inammation [,–]. UV irradiation
enhances the synthesis of proinammatory cytokines in the
skin tissue and stimulates the inltration and activation of
Evidence-Based Complementary and Alternative Medicine
200
300
400
500
600
##
0
100
No UV-B Vehicle Manuka oil
*
TNF-(g/g tissue protein)
(a)
200
300
400
500
600
700
##
0
100
No UV-B Vehicle Manuka oil
**
IL-1(g/g tissue protein)
(b)
UV-B
10% manuka oil
No UV-B control Veh ic le only
(c)
30
40
50
60
##
0
10
20
No UV-B Vehicle Manuka oil
**
Number of CD163+macrophages/field
(d)
F : Manuka oil inhibits UV-B-induced production of inammatory cytokines and inltration of macrophages. e amount of TNF-𝛼
(a) and IL-𝛽(b) were estimated using ELISA in the total protein extract of dorsal skin biopsies obtained at the end of the experiment from
thenoUV-Birradiationcontrolgroup(noUV-B),vehicleonlygroup(vehicle),andthegroupthatreceived%manukaoil(manukaoil),
respectively. Data represent mean ±SEM (𝑛=6per group). ##: signicantly dierent from no UV-B control group (𝑃 < 0.01). ∗and ∗∗:
signicantly dierent from vehicle only group (∗∗𝑃< 0.01 and ∗𝑃< 0.05). (c) Representative images (×) from immunohistochemical
staining of inltrated macrophages in dorsal skin biopsies obtained at the end of the experiment. An example of stained macrophage was
denotedbyaredarrow.Scalebar:𝜇m. (d) Average values ±SEM of macrophage numbers counted per eld were represented as a bar
graph (𝑛=6per group).
inammatory cells such as neutrophils and macrophages in
the skin [,]. UV irradiation of broblasts activates AP-
andNF-𝜅B to induce the production of proinammatory
cytokinessuchasIL-𝛽and IL- [,]. ese cytokines
thenstimulateinammatorycellssuchasneutrophilsand
macrophages in the skin to produce IL-𝛽and TNF-𝛼.
Eicosanoids such as the prostaglandins and leukotrienes
also play a role in provoking skin inammation and their
generation is stimulated under oxidative stress [].
Skin inammation causes premature skin aging because
it can increase the expression of MMPs and damage cellular
and molecular integrity of the dermis and the epidermis
Evidence-Based Complementary and Alternative Medicine
[]. Accordingly, inhibition of UV-irradiation induced-
inammatory responses is important to protect skin from
photoaging. In fact, inhibition of skin inammation by
topical application of anti-inammatory agents attenuates
photoaging []. erefore, we suggest that the inhibitory
activity of manuka oil against skin inammation is crucial
for its antiphotoaging ecacy. Further studies would be
necessarytoidentifythechemicalcomponentsinmanukaoil
that exert anti-inammatory activity. We propose antioxidant
chemicals in manuka oil to be responsible for the anti-
inammatory activity of the oil. To support this hypothesis,
EGCG, a plant antioxidant compound inhibited inltration
of inammatory cells in UV-irradiated mice skin []. It is
also possible for the sesquiterpene compounds present in the
manuka oil to confer an anti-inammatory eect. ere have
been multiple reports suggesting sesquiterpene hydrocarbons
to possess anti-inammatory activity [–].
A recent report indicated that manuka oil possesses a
strongantimicrobialactivityagainstPropionibacterium acnes,
whichsuggeststhatmanukaoilcouldbeeectiveagainstacne
[]. Along with this, our work suggests that manuka oil is
indeed useful in skin care and functional cosmetology.
5. Conclusion
We provide evidence that manuka oil can attenuate cutaneous
photoaging in a controlled study with the help of UV-B-
irradiated hairless mouse model. We suggest antioxidant
chemicals and sesquiterpene compounds in manuka oil to
underlie such an eect. erefore, our work supports that
manuka oil can be used in the formulation of skin care and
functional cosmetic products.
Conflict of Interests
e authors declare that there is no conict of interests in the
current study.
Authors’ Contribution
Oh Sook Kwon and Seung Hee Jung contributed equally to
this work.
Acknowledgments
is study was supported by Fusion Technology Develop-
mentProgramthroughtheSmallandMediumBusiness
Administration (SD) and KIST (E) Grants.
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