Effect of German chamomile oil application on alleviating atopic dermatitis-like immune alterations in mice

Abstract

Historically, German chamomile (GC) oil has been used for treatment of skin disorders. BALB/c mice were sensitized twice a week with 100 microL of 1% 2,4-dinitrochlorobenzene (DNCB) and challenged twice the following week with 100 microL of 0.2% DNCB for atopic dermatitis induction. Thereafter, 3% GC oil was applied daily (70 microL, 6 times week) on the dorsal skin for 4 weeks. Saline or jojoba oil was used for the control mice. Blood was collected after second DNCB challenge, and at 2 and 4 weeks after initiating oil application. Serum IgE levels were significantly lowered in the GC oil application group at the end of the 4-week application period. The GC oil application for 4 weeks resulted in reduction in serum IgG1 level compared with that after 2-week application. The GC oil application group showed a significantly lower serum histamine level than the control group 2 weeks after oil application. Scratching frequency of the GC oil application group was significantly lower than either control groups. This study is to demonstrate GC oil's immunoregulatory potential for alleviating atopic dermatitis through influencing of Th2 cell activation.

Full text

JOURNAL OF
Veterinary
Science
J. Vet. Sci. (2010), 11(1), 35
󰠏
41
DOI: 10.4142/jvs.2010.11.1.35
*Corresponding author
Tel: +82-53-580-5931; Fax: +82-53-588-5233
E-mail: yckim@kmu.ac.kr
Effect of German chamomile oil application on alleviating atopic
dermatitis-like immune alterations in mice
Soon-Hee Lee
1
, Yong Heo
2
, Young-Chul Kim
3,
*
1
Department of Beauty Art, Howon University, Gunsan 573-718, Korea
2
Department of Occupational Health, Catholic University of Daegu, Gyeongsan 712-702, Korea
3
Department of Public Health, Keimyung University, Daegu 704-701, Korea
Historically, German chamomile (GC) oil has been used for
treatment of skin disorders. BALB/c mice were sensitized
twice a week with 100
μ
L of 1% 2,4-dinitrochlorobenzene
(DNCB) and challenged twice the following week with 100
μ
L
of 0.2% DNCB for atopic dermatitis induction. Thereafter,
3% GC oil was applied daily (70
μ
L, 6 times week) on the
dorsal skin for 4 weeks. Saline or jojoba oil was used for
the control mice. Blood was collected after second DNCB
challenge, and at 2 and 4 weeks after initiating oil application.
Serum IgE levels were significantly lowered in the GC oil
application group at the end of the 4-week application period.
The GC oil application for 4 weeks resulted in reduction
in serum IgG1 level compared with that after 2-week
application. The GC oil application group showed a
significantly lower serum histamine level than the control
group 2 weeks after oil application. Scratching frequency
of the GC oil application group was significantly lower
than either control groups. This study is to demonstrate
GC oil’s immunoregulatory potential for alleviating atopic
dermatitis through influencing of Th2 cell activation.
Keywords:
atopic dermatitis, cytokine, German chamomile oil,
IgE, mice
Introduction
In general, atopic dermatitis occurs mainly from
dysregulation in helper T (Th) cell reactivities [1,4]. The
Th cell is classified into two types, types 1 and 2, depending
on type of cytokine secreted by the cell and immunoregulatory
functions driven by the secreted cytokine [11,22,29]. Any
disruption of the balance between these two types of Th
cells may cause a variety of immunological diseases, such
as allergic asthma and allergic rhinitis. Atopic dermatitis is
an immunologic disease induced from an imbalance
favoring type-2 helper T (Th2) cell [3].
Interleukin-4 (IL-4) production from Th2 cells, mast
cells, or other immune components cells is known to
trigger isotype-switching to IgE and IgG1 in B cells [18].
Evaluation of IL-4 production from splenic T cells is a tool
for investigating the predominance of Th2 reactivity [13].
Hyper IgE production is a hallmark of atopic dermatitis in
humans [1,4], and well documented in transgenic mice or
NC/Nga mice suffering from atopic dermatitis-like skin
lesions [8,26,33]. IFNγ from type-1 helper T (Th1) cells,
natural killer cells, other immune cells induces isotype-
switching to IgG2a in mice [17,31]. Therefore, analysis of
the serum IgG1 or IgG2a levels could reflect the alteration
of homeostasis between type-1 and type-2 immune
responses. Since histamine is immediately released
following allergen exposure, increase in serum histamine
level is often considered as a parameter for diagnosing the
onset of allergic diseases. Previous studies also showed
that eosinophils play an important role in atopic dermatitis,
characterized by elevated blood eosinophil counts [17,30].
Atopic dermatitis is a chronic skin disorder and has been
medically treated using steroids, antihistamines,
immunosuppressive agents, and other medications. But
many studies have reported that long-term use or even
abuse of these agents may cause various side effects, so
relevant recent studies have focused upon complementary
therapies based on alternative medicine [9,28].
Aromatic oils are natural essential oils consisting of
volatile organic compounds containing original scents and
therapeutic substances. It is well known for positive effects
in insomnia, relief from anxiety and stress, recovery from
fatigue, muscular relaxation, anti-histaminic or anti-allergic
actions [5,7,27]. German chamomile (GC) oil, an aromatic
oil, has been reported to help relieve physical or mental
stress or fatigue through its sedative or alleviating effects
along with fruity scent, is also known to be effective in the

36 Soon-Hee Lee et al.
Fig. 1. Macroscopic photograph of atopic dermatitis mouse skin
j
ust after induction by the application of 2,4-dinitrochlorobenzen
e
(DNCB) (A). Saline application after atopic dermatitis inductio
n
as the control (B), jojoba oil application after atopic dermatitis
induction as the vehicle (C), and German chamomile (GC) oil
application after atopic dermatitis induction as the experiment (D).
B, C and D are macroscopic photographs at 4 week after various
application.
treatment of dry and itchy skin [23]. Historically, GC oil
has been used for the treatment of skin disorders such as
eczema, as it contains three major sesquiterpene constituents
(azulene, bisabolol, farnesene) with anti-inflammatory or
anti-histaminic effects [6,32]. Particularly, it is known that
these effects of GC oil come from α-bisabolol, a substance
that has strong anti- inflammatory effects of all. However,
there have been no scientific studies to date on the possible
effects of GC oil on alleviating atopic dermatitis. Therefore,
this study tested the application of GC oil on BALB/c mice
demonstrating atopic dermatitis-like immune alteration
and skin lesions. The 2,4-dinitrochlorobenzene (DNCB)
skin application model was adopted to induce atopic
dermatitis-like phenomena in mice, which has been reported
as a reliable model demonstrating similar immunologic
and skin alteration as human atopic dermatitis [24].
Materials and Methods
Composition of GC oil
For the experiment, this study used GC oil (Sanoflore,
France) as 100% pure and natural essential oil tested by
organic product certification organization (ECOCERT,
France) as well as jojoba oil (Desert Whale, USA). Jojoba
oil was used as a control due to its clinical application.
Animals
Forty BALB/c mice (SPF male, 7 weeks old) were
purchased from Daehan Biolink (Korea). Both animal care
and protocol for this study were in accordance with
Institutional Animal Care and Use Committee (USA) and
OECD guideline. Animals fed on unlimited amount of water
and feed throughout whole experiment duration. The
animals were divided into four groups (ten mice each) and
were housed separately in plastic cages per each group. The
normal group was applied with saline throughout atopic
dermatitis induction stage and oil treatment period. The
control group was applied with saline following induction
of atopic dermatitis. The vehicle group was applied with
jojoba oil and the experimental group with 3% GC oil
following atopic dermatitis induction (Fig. 1). Three % GC
oil was selected in accordance to the clinical application.
Induction of atopic dermatitis-like immunologic
and skin disorders
For induction of atopic dermatitis-like immunologic and
skin disorders, DNCB was applied onto mice skin. After
complete removal of dorsal hairs in area of approximately
8 cm
2
, 100 μL of 1% DNCB was applied on their dorsal skin
twice every 3 days for sensitization. In the next week, 100
μL of 0.2% DNCB was applied on their dorsal skin twice
every 3 days for challenge. DNCB was dissolved in a 4 : 1
mixture of acetone and olive oil. As soon as the challenge
was completed, 3% GC oil (diluted in jojoba oil) was applied
once a day (70 μL, 6 times week) on the dorsal skin for 4
weeks. Saline or jojoba oil was applied onto skin of the
control mice. Three hundreds μL of blood was sampled at
the completion of second challenge and 2 weeks after
initiating oil application via periorbital sinus. After the 4-week
oil application period, under ether anesthesia blood sampled
from posterior vena cava was used for immunologic or
hematologic analysis, and skin tissue was sampled and fixed
with 10% neutral buffered formalin solution for histopathologic
examinations, splenic cells were sampled by separation
from the extracted spleen and stored in a freezer after
freezing in liquid nitrogen for cytokine analysis.
Immunologic observation
Measurement of serologic parameters: Serum IgE levels
were determined using a sandwich ELISA method, as
previously described [15]. The levels of serum IgG1 and
IgG2a were also determined using a sandwich ELISA
method, using goat anti-mouse IgG1 and IgG2a capture
antibodies and a peroxidase conjugated anti-mouse IgG
detection antibody [16]. In addition, serum histamine levels
were determined using the IBL histamine ELISA kit (IBL,
Germany) according to the quantification methodology of
Duda et al. [10].
Measurement of IL-4 in splenic cell culture solution:
Splenic cells (5 × 10
5
cells) were stimulated with
immobilized anti-CD3 mAb (5 μg) for 48 h in a CO
2
incubator. The level of IL-4 in culture supernatants was
determined using a sandwich ELISA method prepared by
BD Pharmingen (BD, USA) [15]. The lower limit of

Effects of German chamomile oil on atopic dermatitis 37
Tabl e 1 . Serum level of IgG1 or IgG2a (mg/mL) in mice followin
g
atopic dermatitis induction and test compounds application
Group*
Week after the completion
of the second DNCB
†
challenge
‡
02 4
IgG1
Normal
Control
Vehicle
Experiment
IgG2a
Normal
Control
Vehicle
Experiment
2.34 ± 0.53
a
6.24 ± 0.39
b
7.95 ± 0.84
bc
9.24 ± 0.94
c
0.64 ± 0.06
a
1.30 ± 0.16
b
1.75 ± 0.21
b
1.58 ± 0.17
b
5.13 ± 1.03
a
11.09 ± 1.58
ab
15.90 ± 2.07
ab
19.80 ± 6.03
b
8.88 ± 2.28
a
8.40 ± 2.39
a
16.73 ± 2.03
b
12.09 ± 2.58
ab
6.90 ± 1.15
a
10.18 ± 0.83
ab
15.12 ± 1.63
c
13.75 ± 1.69
bc
8.96 ± 2.41
ab
8.30 ± 1.61
a
15.56 ± 1.52
b
10.89 ± 2.53
ab
*
Normal: No atopic dermatitis induction, Control: Saline applicatio
n
after atopic dermatitis induction, Vehicle: Jojoba oil application afte
r
atopic dermatitis induction, Experiment: GC oil application after
atopic dermatitis induction.
†
DNCB: 2,4-dinitrochlorobenzene.
‡
Th
e
results are expressed as means ± SE (n = 10).
a,b,c
Values with differen
t
superscripts in the same column are significantly different (p ＜ 0.05).
Fig. 2. GC oil-mediated suppression of IgE hyperproduction.
Serum IgE levels were measured at the completion of second DNCB
challenge, 2 and 4 weeks after initiating dermal application of tes
t
compounds. The results are expressed as means ± SE.
a,b,c
Va lu es
with different superscripts are significantly different (p ＜ 0.05).
Fig. 3. Changes in serum histamine levels of the mice sensitized an
d
challenged with DNCB followed by 4-week dermal application o
f
test compounds. The results are expressed as means ± SE.
a,b
Va l u e s
with different superscripts are significantly different (p ＜ 0.05).
detection was 5 pg/mL for IL-4.
Behavioral observation
Behaviors of mice were monitored according to
observation methodology of Kobayashi et al. [20]. The
frequency of scratching occurring on facial or dorsal skins
was counted with a 30-min visual observation on the first
day and on the 21st day after GC oil application.
Data analysis
All data collected in this study were statistically processed
via SPSS WIN (SPSS, USA). ANOVA was used to test for
differences among the groups. Post-hoc analysis was
carried out using the Duncan’s multiple range test to test
for significant difference among the means (p ＜ 0.05).
Results
GC oil-mediated suppression of IgE hyperproduction
Mice at the completion of 0.2% DNCB second challenge
demonstrated significantly higher levels of serum IgE
(approximately 4.4 times) than the normal group (Fig. 2),
implying a successful induction of atopic dermatitis-like
immune alteration. Serum IgE levels were significantly
downregulated in the experimental group after application
of GC oil for 4-weeks, while the saline-applied control or
the jojoba oil-applied vehicle groups still demonstrated
significantly upregulated IgE levels compared to the
normal mice.
Modulation of serum IgG1 level following GC oil
application
Levels of IgG1 and IgG2a were substantially increased
following DNCB sensitization and challenge compared to
the normal mice (Table 1). Application of GC oil for 4
weeks resulted in a reduction of approximately 31% (13.75
mg/mL) in IgG1 levels compared to 19.80 mg/mL after
2-weeks of GC oil application. There were no significant
differences in the levels of IgG1 in saline-applied control
or jojoba oil-applied vehicle groups between at 2 week and
at 4 week following atopic dermatitis induction. Meanwhile,
persistent application of GC or jojoba oil did not lead to
any changes in IgG2a levels when compared between 2
and 4 weeks following the induction of atopic dermatitis.

38 Soon-Hee Lee et al.
Fig. 4. Effect of GC oil application on production of interleukin-4
(IL-4) from splenic T cells. Splenocytes were stimulated with
immobilized anti-CD3 mAb for 48 h. Culture supernatants were
collected for measurement of IL-4. The results are expressed as
means ± SE.
Fig. 5. Decreased scratching frequency in GC oil-treated mice.
Frequency of scratching on facial and back skin was measure
d
for 30 min one day after the second DNCB challenge (day 1) an
d
the 21st day after initiating GC oil application (day 21). The
results are expressed as means ± SE.
a,b,c
Values with different
superscripts are significantly different (p ＜ 0.05).
Tabl e 2 . Changes in number of inflammatory cells in GC oil-treate
d
mice skin at 4 week after various application following atopic
dermatitis induction
Group Neutrophils
*
Lymphocytes
*
Normal
Control
Ve hi cl e
Experiment
1.20 ± 0.13
a
1.90 ± 0.23
b
1.50 ± 0.17
ab
1.30 ± 0.15
a
2.00 ± 0.26
a
4.20 ± 0.42
c
3.20 ± 0.25
b
2.30 ± 0.26
a
*
The results are expressed as means ± SE (n = 10) of microscopic
observation at ×200 magnification.
a,b,c
Values with different
superscripts in the same column are significantly different (p < 0.05).
N
ormal: No atopic dermatitis induction, Control: Saline applicatio
n
after atopic dermatitis induction, Vehicle: jojoba oil application afte
r
atopic dermatitis induction, Experiment: GC oil application afte
r
atopic dermatitis induction.
Effect of GC oil application on modulation of serum
histamine level
Histamine levels were significantly higher in all mice
groups exposed to DNCB compared to the normal mice
(Fig. 3), indicating a successful induction of atopic
dermatitis-like alterations of the immune system. Two
weeks after GC oil application, the GC oil group (18.45
ng/mL) showed significantly lower (approximately 51%)
serum histamine level than the saline-applied control
(37.43 ng/mL), and also lower (approximately 40%) than
the jojoba oil-applied vehicle (30.60 ng/mL) group.
However, no further downregulation of histamine levels
was found 4 weeks after the start of treatment.
Effect of GC oil application on production of IL-4
from splenic T cells
Four weeks of GC oil application (10.4 pg/mL) resulted
in lower (approximately 50%) IL-4 production with no
statistical significance (p ＞ 0.05) in splenic cell culture
supernatants compared to the saline treated control mice
(20.7 pg/mL) (Fig. 4). These findings imply that GC oil
controls the production of IL-4 from Th2 cells, contributing
to controlling IgE and IgG1 generation from B cells.
Decreased scratching frequency in GC oil-applied mice
The frequency of scratching on facial and back skin was
observed for 30-min on one day after the second DNCB
challenge (day 1) and on the 21st day after initiating GC oil
application (day 21). It was found that the GC oil group (44
times) and jojoba oil group (65 times) showed significantly
lower (approximately 45% and 19%, respectively) scratching
frequency than the saline treated control group (80 times)
at day 21 (Fig. 5). Furthermore, scratching frequency of the
GC oil group was significantly lower (approximately 32%)
than the jojoba oil vehicle group.
Decreased inflammatory cell infiltration in GC
oil-applied mice skin
Numerous leukocytes were infiltrated into the dermis and
epidermis was thickened in the control mouse skin
compared to the normal mouse skin, while inflammatory
cell infiltration, such as neutrophils and lymphocytes, and
hyperplasia of the epidermis were significantly (p ＜ 0.05)
reduced in the experimental mouse skin (Table 2 and Fig.
6). In this study, it was found that application of GC oil for
4 weeks resulted in significantly lower leukocytes in the
peripheral blood than the saline treated control mice, and
showed similar values to the normal mice (Table 3). The
number of neutrophils, lymphocytes, monocytes, eosinophils
and basophils of GC oil application for 4 weeks showed
significantly (p ＜ 0.05) lower levels than the saline treated

Effects of German chamomile oil on atopic dermatitis 39
Fig. 6. Histophatological findings at 4-week after various applicatio
n
following atopic dermatitis induction. No atopic dermatitis inductio
n
as normal mice (A), saline application following atopic dermatitis
induction as control mice (B), jojoba oil application following
atopic dermatitis induction as vehicle mice (C), and GC oil
application following atopic dermatitis induction as experimenta
l
mice (D). H&E stain, ×100.
Tabl e 3 . Changes in number of leukocytes at 4 week after various application following atopic dermatitis induction
Items
Group
Normal Control Vehicle Experimental
Total WBC
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
3.39 ± 0.33
a
1.03 ± 0.12
a
2.01 ± 0.17
a
0.29 ± 0.05
a
0.04 ± 0.01
a
0.02 ± 0.01
ab
5.21 ± 0.32
b
1.53 ± 0.13
b
2.95 ± 0.19
b
0.59 ± 0.04
b
0.10 ± 0.03
b
0.04 ± 0.01
b
3.87 ± 0.26
a
1.16 ± 0.06
a
2.32 ± 0.20
a
0.34 ± 0.03
a
0.04 ± 0.01
a
0.02 ± 0.01
a
3.46 ± 0.19
a
1.05 ± 0.04
a
1.99 ± 0.15
a
0.34 ± 0.02
a
0.05 ± 0.01
a
0.02 ± 0.01
ab
The results are expressed as means ± SE (n = 10).
a,b
Values with different superscripts in the same row are significantly different (p ＜ 0.05).
Unit: ×10
9
/L.
control mice by 31, 33, 42, 50 and 50%, respectively (Table 3).
Discussion
The present study was undertaken to evaluate the efficacy of
GC oil alleviating atopic dermatitis-like immune alterations
in mice. Even though GC oil has been historically used for
the treatment of pruritic skin disorder [2,6,12,32], there has
been no scientific study investigating GC oil on the
treatment or prevention of atopic dermatitis. Our study is
the first to demonstrate GC oil’s immunoregulatory potential
for alleviating atopic dermatitis through influencing of Th2
cell activation. Overall, we found that GC oil possessed an
ability to influence Th2 cell activation involved with the
onset or progression of atopic dermatitis, in that dermal
application of GC oil resulted in the suppression of IgE or
IgG1 over-production, downregulation of IL-4 production
from Th2 cells, and demonstrated a reduction in histamine
release. In addition, frequent scratching due to itchy
sensations was alleviated following GC oil application
onto the damaged skin involved with the pathogenesis of
atopic dermatitis.
Histamine release from activated mast cells or basophils
is a hallmark of the occurrence of allergic diseases such as
atopic dermatitis, and cause itching, increased vascular
permeability, and the wheal and flare response of immediate
hypersensitivity [1]. Pruritus is a symptom found in a
variety of skin disorders like atopic dermatitis and contact
allergic dermatitis. For atopic dermatitis cases, it has been
reported that scratching due to itchy sensations causes skin
damage, promotes inflammation and thereby further
aggravates pruritus [1,4,21,34,35]. Therefore, it is important
to reduce itchy sensations and scratching frequency to prevent
aggravation of skin lesions due to pruritic disorders and
improving quality of life.
Kobayashi et al. [19,20] reported that the oral administration
of GC extract combined with histamine receptor antagonists
was more effective for the suppression of scratching
behavior in mice than administration of histamine receptor
antagonists alone. GC oil may inhibit the binding of
histamine to its receptor, which will eventually control
histamine mediated clinico-pathologic effects such as itching
[20]. Concerning our results showing the downregulation
of serum histamine levels 2 weeks after the start of GC oil
application following atopic dermatitis induction, this
compound may also exhibit its anti-histamine effect by
directly suppressing histamine release from mast cells
through downregulation of IL-4 production from Th2 cells
and following by suppression of IgE or IgG1 over-production.
The most important aspect to be discussed is GC oil’s
systemic effect on the modulation of the in vivo type-2
response. The type-2 response becomes predominant when

40 Soon-Hee Lee et al.
differentiation or activation of Th2 cells is preferred, but
development or stimulation of Th1 cells is suppressed
[13,14]. Considering that the skewedness toward type-2
responses is a background mechanism for the occurrence
of atopic dermatitis [1,4,25], dermal application of GC oil
could systematically block the pathogenesis of atopic
dermatitis through correcting the immune homeostasis
skewed in favor of Th2. Considering our experimental
results, it could be concluded that the application of GC oil
on the skin of atopic dermatitis cases could alter production
of IL-4 from Th2 cells and also alter IgE, IgG1 and histamine
production, performing a series of immunoregulatory
functions to alleviate the occurrence or progression of
atopic dermatitis. Hence, it is believed that the application
of GC oil on atopic dermatitis cases will be significantly
meaningful for public health and alternative medicine.
Although it is known that α-bisabolol has the strongest
anti-inflammatory effects of all the constituents of GC oil,
it is not clear which constituent(s) of GC oil contribute to
the GC oil-mediated alleviation of atopic dermatitis-like
immunologic and skin alterations in mice at the moment.
Future studies will be directed towards identifying which
components of GC oil are responsible for its
immunomodulatory effects. Furthermore, whether GC oil
exerts its immunomodulatory effects independently,
sequentially, or concomitantly on each of the immune
component cells involved in the pathogenesis of atopic
dermatitis such as Th, B, and mast cells needs to be
answered. Further investigations are necessary to identify
target immune cells for GC oil-mediated alleviation of
atopic dermatitis-like immunologic and skin alterations.
Acknowledgments
This study was conducted with the support of the research
fund of Howon University, Korea.
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