Targeting Inflammatory Pathways to Reduce Dark Circles and

CO S MET IC S
I N F L A MMAT IO N
H. Chajra, D. Auriol, K. Schweikert, F. Lefevre*
Targeting Inflammatory Pathways to Reduce
Dark Circles and Puffiness
■ Introduction
Dark circles under or around the eyes
(characterized by the darkening of the
eyelids and periorbital skin) are a common cosmetic problem for a large number of people as is often associated with
tiredness, stressed or aged appearance
(1). Various causes can lead to the ap-
pearance of dark circles around the eyes,
including aging with increased skin laxity, post-inflammatory hyperpigmentation secondary to atopic dermatitis or
allergic contact dermatitis (2, 3), or as
a consequence of post-inflammatory
hemodynamic congestion producing
a typical bruising aspect on the lower
eyelids and oedema (3). Oedema is often
Abstract
D
ark circles are a common cosmetic problem for a large number of people. Because, most of the cosmetic products available on the market
targeted either blood micro-circulation or covered the skin hyperpigmentation, we decided to develop a cosmetic ingredient, which targets the
inflammatory, lymphatic and vascular origin of dark circles. In this study the
efficacy of the active ingredient was assessed in vitro and on human volunteers. Our studies have shown the effectiveness of this ingredient to reduce
the dark circles after 15 and 30 days of treatments. The mechanism of action
demonstrated is based on both anti-inflammatory and draining properties.
The active ingredient showed anti-inflammatory effects on keratinocytes by
strongly inhibiting NF-KB pathway-driven IL-8 and CXCL1 releases. The inhibitory effect of the active ingredient was not mediated by a direct inhibition
of NF-KB translocation but by inhibition of the NF-KB activity. This active ingredient improves the drainage function of lymphatic vessels by increasing
the expression of VEGFR3 gene and controls the vascular tone by increasing
the expression of Heme oxygenase-1 gene.
16
due to a malfunction of lymphatic vessels. Histo-pathological changes associated with dark circles are the increase in
melanin content causing hyperpigmentation and the dilation of dermal blood
vessels (4). Periocular »dark circles« fall
among the most difficult complaints
to address by dermatologists. There is
no gold-standard treatment option.
Available dermatological treatments
include bleaching creams, topical retinoid acid, chemical peels, laser therapy,
autologous fat transplantation, injectable fillers, surgery (blepharoplasty), and
chemical peeling. Most of the cosmetic
products available on the market target
either skin lightening, vascular constriction (using caffeine or esculin), or they
use pigments to cover the skin hyperpigmentation.
Our searches targeted the inflammatory, lymphatic and vascular origins of
dark circles. Gallic acid and its derivatives including epigallocatechin gallate
are known for their anti-inflammatory
properties by acting on the inhibition
of the degranulation by neutrophils (5),
decreasing cytokine production (6-9),
reducing immune cell infiltration and
inhibiting lipooxygenase (10). However,
these molecules are well known to be
either instable and/or difficult to formulate in cosmetic products due to their
chemical structures. We have therefore
combined in a water solution a mix of
three stable and water-soluble gallic
acid derivatives: gallic acid glucoside,
epigallocatechin gallate glucoside, and
propyl gallate (patent Libragen company
EP2027279B1 (11)). We have studied in
vitro the activity on the NF- B pathway
of the active ingredient. More precisely
SOFW-Journal | 140 | 4-2014
CO SMET ICS
I NF LA MMAT I O N
the effects of the active ingredient were
evaluated on nuclear factor-kappa B
translocation, and then on the activation of IL-8 and CXCL1 release by keratinocytes. To address the lymphatic
and vascular origins, in vitro activities
of the active ingredient on the lymphatic
factor (VEGFR3) and the vascular factor (Heme Oxygenase -1) were studied.
Finally, to evaluate the efficacy of the
active ingredient to treat dark circles a
clinical investigation was performed on
human volunteers.
■ Materials and methods
The active ingredient (trade name Unisooth EG-28) was composed by 6mM of
gallic acid glucoside (CAS 131579-696), 323µM of epigallocatechin-glucoside
(CAS 236072-19-7), and 117 mM propyl gallate (CAS 121-79-9). Gallic acid
glucoside is the glycosylated form of
gallic acid coming from oak leaves and
epigallocatechin-glucoside is the glycosylated form of epigallocatechin gallate
originating from green tea.
Studies on the inflammatory pathways
NF- B translocation in keratinocytes
Cell cultures and treatments: The normal
human epidermal keratinocytes (NHEK
used at the 3rd passage, Bioalternatives, Gençay, France) were cultured in
96-well plates in culture medium until
subconfluence at 37°C, and 5 % CO2.
20000 cells/well were seeded. The culture medium was a Keratinocyte SFM
supplemented with epidermal growth
factor (EGF) at 0.25 ng/mL, pituitary extract (PE) at 25 µg/ml and gentamycin
at 25 µg/ml. The culture medium was
then removed and replaced by assay
medium containing or not the active
ingredient (at 0.016, 0.08 and 0.4 %) or
the references (NF- B inhibitor III at 5
µM + NF- B inhibitor V at 1 µM) and the
cells were pre-incubated for 2 hours.
The assay medium was a keratinocyteSFM supplemented with gentamycin at
25 µg/ml. After the pre-incubation, the
cells were stimulated by the association
of TNF- + IL-1 (both at 5 ng/ml) and
incubated for 20 minutes. All experimental conditions were performed in
triplicate.
NF- B translocation- in situ immunolabeling: At the end of incubation, culture medium was discarded and the cells
were washed, fixed and permeabilized.
Cells were then labeled with a primary
anti-p65 NF- B antibody (ref 610868;
BD Biosciences, France). The primary antibody was then revealed using a fluorescent secondary antibody (GAM-Alexa
488) and the cell nuclei were labeled with
Hoechst solution (bis-benzimide) in parallel. The acquisition of the images was
performed with the INCell AnalyzerTM
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SOFW-Journal | 140 | 4-2014
17
CO S MET IC S
I N F L A MMAT IO N
1000 (GE Healthcare). Controls without
primary antibody were performed in order to adjust the acquisition parameters
of the camera. 5 photos were taken per
well for the immunolabeling of NF- B.
The labeling was quantified by the measurement of the fluorescence intensity
in cell nuclei (Integration of numerical
data with the Developer Toolbox 1.5, GE
Healthcare software).
NF- B activity study in transformed
human cells
Cell cultures and treatments: Transformed human cells (HT29) were transfected with a NF- B reporter gene plasmid (pNiFty2-SEAP, reference: pNifty2SEAP, Invivogen, Toulouse, France). The
transfected cells (500000/mL) were incubated 24 h with TNF- (20 ng/mL) in
the presence or absence of active ingredient at different concentrations (from
0.01 to 4.36 %). All experimental conditions were performed in quadruplicate.
Assay of NF- B activity: The contact of
TNF- and transfected cells (HT29-NFB -SEAP-Cl25) induced gene expression
of the NF- B pathway as well as SEAP
(secreted Embryonic Alkaline Phosphatase) loaded by the plasmid pNiFty2SEAP. The expression of this gene and
the production of the SEAP enzyme were
directly proportional to the quantity of
cytokine present in the culture media.
The assay of SEAP enzyme is an indicator
of NF- B pathway activation. The assay of the enzyme SEAP was performed
using the Quantie blue reactif (ref repqb, Invivogen, France) according to the
instructions of use provided by the fabricant. The enzyme concentration was
determined by the reading of the absorbance at 650 nm.
IL-8 and CXCL1 released by
keratinocytes
Cell cultures and treatments: The keratinocytes (NHEK) were cultured in 96-well
plates in culture medium for 24 hours
at 37°C, and 5 % CO2. 20000 cells/well
were seeded. The culture medium was
a Keratinocyte SFM supplemented with
epidermal growth factor (EGF) at 0.25
ng/mL, pituitary extract (PE) at 25 µg/
ml and gentamycin at 25 µg/ml. The
medium was then removed and re-
18
placed by assay medium containing or
not (control) the active ingredient or the
references (NF- B inhibitor III at 5 µM
and dexamethasone at 10 µM) and the
cells were pre-incubated for 24 hours.
The assay medium was a keratinocyteSFM supplemented with gentamycin at
25 µg/ml. After the pre-incubation, the
medium was removed and replaced by
assay medium containing or not (control) the active ingredient or the references in presence of the association of
TNF- + IL-1 (both at 5 ng/ml) and the
cells were incubated for 48 hours. A nonstimulated control condition was also
performed in parallel. All experimental
conditions were performed in triplicate.
Quantification of IL-8 and CXCL1 released by NHEK: At the end of incubation,
the quantities of IL-8 (Elisa Development
kit, ref 900-K18, Peprotech, Neuilly sur
Seine, France) and CXCL1 (Ref. DY275,
R&D Systems Europe, Lille, France) in
culture supernatants were measured using ELISA kits according to the supplier’s
instructions.
Data management: Raw data were analyzed with Microsoft Excel software.
The inter-group comparisons were performed by Student’s t-test (for paired
data). The significance was judged as followed. *p <0.05; **p <0.01 and ***p< 0.001.
All reported data are expressed as mean
± sem. The standard error of the mean
(sem) is calculated as the standard deviation (sd) divided by the square root of
sample size (sem = Sd/ n).
Studies on the lymphatic and
vascular markers
The effects of the active ingredient at
0.04 and 0.2 % were studied on gene expression of two proteins involved in vessel functions using RT-qPCR technology.
Cell cultures and treatments: Human dermal microvascular endothelial
cells (HMVEC, Bioalternatives, Gençay,
France) were cultured in culture medium
for 48 hours (10000 cells per well). The
medium was then removed and replaced
with assay medium. After 24 hours of
culture, medium was replaced with assay
medium containing or not (control) the
active ingredient then cells were incubated for 24 hours. All experimental
conditions were performed in triplicate. At the end of the incubation, cells
were washed in phosphate buffered
saline (PBS; Life Technologies) solution
and immediately frozen at -80°C until
mRNA extraction.
RT-qPCR and data management: Extracted mRNA from HMVEC was analyzed on a
customized PCR array containing target
genes (VEGFR-3 and Heme Oxygenase 1)
and including 3 housekeeping genes
(glyceraldehyde-3-phosphate dehydrogenase, actin beta and Ribosomal protein S28). Primer sequences used were
Homo sapiens VEGFR-3 (NM_182925):
sense-TGTCCTACGATGCCAGCCAGTG
antisense-TTGAGGTGGTTGCCGATGTGAATG
and Homo sapiens Heme Oxygenase 1
(NM_002133):
sense-TCCGATGGGTCCTTACACTC
antisense- ATTGCCTGGATGTGCTTTTC
Total RNA was extracted using »TriPure
Isolation Reagent« kit (Roche Applied
Science). The amount and quality of RNA
were evaluated using a lab-on-a-chip
Bioanalyzer (Agilent technologies). Potential contaminant traces of genomic
DNA were removed using the DNA-free
system (Ambion by Life Technologies).
The reverse-transcription of mRNA was
conducted in presence of oligo (dT) and
SuperscriptTM II reverse-transcriptase
(Life Technologies). Quantification of
cDNA was performed using NanoVue
Plus (GE Healthcare) and adjustment of
cDNA at 5 ng/µl. The PCRs (Polymerase
Chain Reactions) were performed using
the LightCycler® system (Roche Diagnostic, France) as described originally
by Wittwer et al (12). The incorporation
of fluorescence in amplified DNA was
continuously measured during the PCR
cycles. This resulted in a »fluorescence
intensity« versus »PCR cycle« plot allowing the evaluation of a relative expression (RE) value for each marker. The RE
value was expressed in arbitrary units
according to the formula:
1/2number of cycles x 106
The relative expression calculated was
normalized to the three housekeeping
genes and to untreated cells (control).
SOFW-Journal | 140 | 4-2014
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Table 1 was used for the interpretation
of the effect.
Clinical study on human volunteers
A double blind and placebo-controlled
clinical evaluation was carried out on
25 volunteers (female Caucasian subjects older than 18 years) following
half-face method. The active ingredient
(INCI composition: water, propyl gallate, gallyl glucoside, epigallocatechin
gallatyl glucoside) was incorporated in
a vehicle formula (INCI composition:
aqua, octyldodecyl neopentanoate, octyldodecyl, octyldodecanol, myristyl
myristate, acrylates/C10-30 alkyl acrylate crosspolymer, sodium hydroxide,
phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben,
isobutylparaben). The placebo was made
of the formula without the active ingredient. The efficacy of a cosmetic product
at 3 % on the reduction of the visibility
of dark circles and bags/puffiness under
eyes was studied after 15 and 30 days of
daily use. The active and placebo were
applied twice a day (morning and evening) around the eyes then the area is
gently massaged.
Evaluation of the reduction of the characteristic colors of the shadows under
the eyes: The measurement of the color
of the eye circles was done by means of
a spectrophotometer/colorimeter CM700d (Konica Minolta). The instrument
was able to evaluate the color according to a standard method defined by
the International Lighting Commission
in 1976 (CIE lab model). The dark color
(seen in a dark circle) was represented
by contribution of two colors (red and
blue). The red color was linked to (a)
parameter and the blue color was linked
to (b) parameter. The variation of these
Relative expression ( % of control)
>300 %
>200 % and <300 %
>30 % and <50 %
<30 %
Clinical classification dark circles at D15 and D30
no variation
1
slight improvement
2
moderate improvement
3
remarkable improvement
4
Table 2 Clinical scorage for dark circles.
Fig. 1 NF- B translocation studies (immuno-fluorescence staining).
two parameters ( a and b) was followed by the spectrophotometer. A decrease on the (a) value and an increase
of the (b) value indicates the resorption
of the black color.
Evaluation of the reduction of bags/
puffiness: The measurement of the bags/
puffiness was done by means of a PRIMOS Optical 3D Skin Measuring Device
(GFMesstechnik, GmbH). The optical 3D
measuring devices PRIMOS was based on
digital stripe projection. The technique
Classification of the effect
strong stimulation
stimulation
inhibition
strong inhibition
Table 1 Classification of the effect of the active ingredient.
20
Score
allowed to take a high resolution image
of the skin, take a 3 dimensional image
and analyze by means of image analysis
the informations of the 3D image.
Clinical evaluation: The dermatologist
evaluated the visibility of eye dark circles
in accordance with the clinical scores
reported in Table 2.
Digital Photographs: Digital photographs
were taken before and after product use
by means of a professional digital reflex
camera (Nikon D300, Nikon corporate,
Japan) equipped with a macro lens (AF-S
Micro Nikkor 60 mm f/2.8 G ED, Nikon
corporate, Japan) and a flash system (kit
R1C1, Nikon corporate, Japan).
Statistical analysis: The inter-group comparisons were performed by Student’s ttest (for paired data). The significance was
judged as followed: ns (not significant) p
>0.05, *p <0.05; **p <0.01 and ***p< 0.001.
SOFW-Journal | 140 | 4-2014
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■ Results
tent inhibitory effect at the second and
highest concentrations (95 and 99 % of
inhibition, respectively).
Anti-inflammatory studies
Effect on NF- B translocation in NHEK
(Fig.1): Treatment of NHEK by TNF+IL-1 (stimulated control) induced
the nuclear translocation of NF- B from
cytosolic to nuclear compartment. The
reference, i.e. the association of NF- B
inhibitor III + NF- B inhibitor V, inhibited strongly the TNF- +IL-1 -induced
NF- B nuclear translocation. The active
ingredient at all tested concentrations,
did not modify TNF- +IL-1 induced
NF- B nuclear translocation.
Effect on NF- B activation (Fig. 2 and
Table 3): Treatment with increasing concentrations of active ingredient inhibited up to -85 % of the NF- B activity in
human cells induced by TNF- .
At this stage, we have demonstrated
that the active ingredient interacts with
NF- B pathway, by inhibiting the NFB activity and not by inhibiting NF- B
nuclear translocation.
Effect on IL-8 release by NHEK (Fig. 3
and Table 4): In non-stimulated basal
conditions, no IL-8 release was detected in NHEK cultures. The activation of
NHEK by the association of TNF- +
IL-1 resulted in significant IL-8 release (~ 1735 pg/ml). The references
NF- B inhibitor III and dexamethasone
both importantly inhibited the TNF+ IL-1 - induced IL-8 release by NHEK
(66 and 59 % of inhibition, respectively). The active ingredient, tested at
0.016 %, 0.08 % and 0.4 %, presented
a dose- dependent inhibitory effect by
presenting a moderate inhibitory effect on IL-8 release by activated NHEK
at the lowest concentration (55 % of
inhibition) and a very strong and po-
Effect on CXCL1 release by NHEK (Fig. 4
and Table 5): In non-stimulated basal
conditions, CXCL1 release by NHEK was
Fig. 2 Regulation of NF- B activity by the active ingredient.
Fig. 3 IL-8 release by NHEK.
Criteria
Concentration in active ingredient ( %)
detected (~ 564 pg/ml). The activation
of NHEK by the association of TNF+ IL-1 resulted in significant increase
of CXCL1 release (~ 1689 pg/ml). In
presence of the reference NF- B inhibitor III, CXCL1 release was equivalent to the CXCL1 release observed in
TNF(20 ng/mL)
Active ingredient + TFN- (20 ng/mL)
4.36
2.18
1.09
0.55
0.27
0.14
0.07
0.03
0.02
0.01
0.04±0.001
0.042±0.001
0.05±0.001
0.06±0.005
0.085±0.01
0.104±0.008
0.186±0.030
0.240±0.035
0.203±0.029
0.246±0.033
Variation in absorbance (VS TNF- alone
0.29
0.29
0.28
0.27
0.24
0.22
0.14
0.09
0.12
0.08
% of inhibition of NFKB pathway
87.66
87.17
84.75
81.75
73.86
68.01
43.09
26.44
37.78
24.69
Absorbance at 650 nm (mean±sem)
0.327±0.040
Table 3 Assay of NF- B activity (Absorbance measurement).
SOFW-Journal | 140 | 4-2014
21
CO S MET IC S
IN F L A MMAT IO N
basal conditions. Thus NF- B inhibitor
III almost totally inhibited the TNF- +
IL-1 -induced CXCL1 release (99 % of
inhibition). The reference dexamethasone induced a significant but only a
moderate inhibitory effect on CXCL1
release by activated NHEK (37 % of inhibition). The active ingredient at the
lowest test concentration, significantly
but moderately inhibited CXCL1 release
by activated NHEK. At the second and
highest test concentrations, this compound presented a very potent inhibitory effect (134 and 136 % of inhibition, respectively), since the CXCL1 release was below the basal release level.
Lymphatic and vascular marker studies
Effect on VEGFR-3 and on Heme Oxygenase 1 (HMOX-1) mRNA expressions
(Fig. 5): The active ingredient tested at
0.04 % and 0.2 % stimulated strongly
the transcription of the VEGFR-3 gene.
The relative expressions were respectively 540 and 775 % in comparison to the
mean of the three housekeeping genes
used. For the gene HMOX-1, only the
concentration at 0.2 % induced a significant stimulation effect (294 %).
The active ingredient tested at 0.2 %
stimulated strongly the transcription of
the VEGFR-3 and HMOX-1 genes.
Clinical study
Clinical effect on dark circles: The use of
the product containing active ingredient
reduced significantly the dark colour of
the dark circles under the eyes as shown
by the decrease of a parameter and increase of b parameter (Fig. 6 and Table 6)
at D15 and D30. The effect of active
ingredient on the monitored parameters was greater than those recorded
for placebo. The difference between two
Fig. 4 CXCL-1 release by NHEK.
Treatment
Test compound
Stimulated control
(IL-1 +TNF- )
Basic data
Normalized data
Concentration
IL-8 (pg/ml)
Mean IL-8 (pg/ml)
sem (pg/ml)
% Stimulated control
sem ( %)
5 ng/ml
1602.9
1224.8
2376.9
1734.9
339.1
100
20
<31.3
<31.3
0.0
<2
0
Non-stimulated
control
p
Inhibition ( %)
sem ( %)
p
0
20
**
100
0
**
NF- B inhibitor III
5 µM
510.8
619.4
688.4
606.2
51.7
35
3
*
66
3
*
Dexamethasone
10 µM
624.5
663.9
899.5
729.3
85.9
42
5
*
59
5
*
0.016 %
969.9
715.1
705.8
796.9
86.5
46
5
ns
55
5
ns
0.08 %
113.3
117.6
113.1
114.7
1.5
7
0
**
95
0
**
0.4 %
34
48.5
44.8
42.4
4.4
2
0
**
99
0
**
Active ingredient
Threshold for statistical significance ns: p>0.05, Not significant; *p: 0.01 to 0.05, Significant; **p:0.001 to 0.01, Very significant
Table 4 IL-8 release by NHEK.
22
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CO S MET IC S
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products was statistically significant at
all experimental times. The efficacy of
the product was also confirmed by both
the clinical evaluation carried out by the
dermatologist (Table 7) and the majority of volunteers participating in the
study (66.7 % of the volunteers found a
moderate to remarkable improvement of
their dark circles in comparison to only
41.7 % with placebo).
Clinical effect on bags/puffiness of the
active ingredient:
The use of the active ingredient reduced the bags/puffiness volume at
D15 and D30 (Table 8 and Fig. 7). The
effect of the active ingredient on the
monitored parameters was greater
than those recorded for placebo, which
did not show any evolution upon time
(table 8): -10 % of puffiness reduction
with a very high statistical significance
(p<0,001).
This measured effect was visible on the
clinical images taken during the study,
on which a clear reduction of the bags
(puffiness) under eyes as well as a brightening of the dark color can be seen. One
must notice that such a reduction was
quite good as it is well known that the
reduction of puffiness and dark circles
on aged volunteers is more difficult than
on young ones due to a reduce biological
metabolism.
■ Discussion
Fig. 5 Relative gene expression of VEGFR3 and HMOX-1 genes (RT-qPCR analysis)
CXCL-1 release by NHEK.
Treatment
Test compound
Stimulated control
(IL-1 +TNF- )
The active ingredient showed anti-inflammatory like effects on NHEK by strongly
inhibiting the NF- B pathway-driven IL-8
and CXCL1 releases. However, the inhibitory effect of the active ingredient was
not mediated by a direct inhibition of
NF- B translocation but by the inhibition the NF- B activity. These results are
Basic data
Normalized data
Concentration
IL-8 (pg/ml)
Mean IL-8 (pg/ml)
sem (pg/ml)
% Stimulated control
sem ( %)
5 ng/ml
1536.4
1674.1
1858.5
1689.6
93.3
100
6
785.0
497.1
410.8
564.3
113.1
33
7
Non-stimulated
control
p
Inhibition ( %)
sem ( %)
p
0
8
**
100
10
**
NF- B inhibitor III
5 µM
736.7
514.1
476.9
575.9
81.1
34
5
***
99
7
***
Dexamethasone
10 µM
1234.3
1126.8
1448.6
1269.9
94.6
75
6
*
37
8
*
0.016 %
1144.3
1189.8
1438.3
1257.5
91.3
74
5
*
38
8
*
0.08 %
177.4
<156.3
204.4
<179.4
13.9
<11
1
***
>134
1
***
0.4 %
<156.3
<156.3
<156.3
156.3
0
<9
0
***
>136
0
***
Active ingredient
Threshold for statistical significance ns: p>0.05, Not significant; *p: 0.01 to 0.05, Significant; **p:0.001 to 0.01, Very significant; ***p<0.001 extremely significant
Table 5 CXCL1 release by NHEK.
24
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CO S MET IC S
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Fig. 6 Effect of the active ingredient and placebo on a and b parameters.
Fig. 7 Digital photography of puffiness and dark circles at D0 (before treatment)
and D30 (after treatment with active ingredient)
Treatment
Test compound Concentration
Placebo
Active
3%
Ingredient
Test compund
Concentration
Placebo
Active
3%
Ingredient
consistent with data from other authors
(7, 13). Inhibition of the NF- B pathway
was already described for epigallocatechin-3 gallate (EGCG) (7) and gallic
acid derivatives (9, 13). EGCG and gallic
acid derivatives are component of the
active ingredient. It has been shown in
the literature that for EGCG, the inhibition is based on the down-regulation of
p-I B , p65, p-p65 (7). For gallic acid
derivatives the inhibition was reported
to be based on the significant inhibition
of the nuclear translocation of p65, (a
subunit of nuclear factor- B (NF- B)),
on the inhibition of the phosphorylation
of IkB, I B kinase and p65, the downregulation of the expression of NF- Bregulated inflammatory cytokines (IL-8,
TNF- , IL-1 ).
This study has also shown that the active
ingredient has a role in vessels functions
modulation due to its regulation effect
observed on two VEGFR3 and HMOX-1
genes. Indeed, the active ingredient
improves the drainage function of lymphatic vessels by increasing the expression of VEGFR3 gene; while controling
the vascular tone by increasing the expression of the Heme oxygenase-1 gene.
The crucial role of VEGFR3 signalling
pathway in lymphatic drainage was revealed by the studies of Mendola et al
(14) showing that familial lymphedema
are due to mutation in VEGFR3 gene.
Hagura et al (15) showed that blockage by antibody of VEGFR3 inhibited the
drainage function of the lymphatic system (oedema is persistent) and increase
the inflammation. VEGFR3 signalling
pathway is fundamental for the mediation of drainage function and in the control of skin inflammation. On the other
D0
a (mean±sem)
(3.39±0.195)
D15
a (mean±sem)
(3.12±0.179)
D30
a (mean±sem)
(3±0.0.185)
(3.28±0.176)
(2.81±0.162)
(2.67±0.0.147)
a (mean±sem)
(-3.05±0.272)
a (mean±sem)
(-3.02±0.293)
a (mean±sem)
(-2.8±0.276)
(-2.97±0.200)
(-2.62±0.185)
(-2.42±0.174)
% of variation
D15 versus D0 D30 versus D0
(-7.2 %)**
(-10 %)**
(-14.2 %)***
(-17.7 %)***
D15 versus D0
2.3 % ns
D30 versus D0
8.9 % **
11.5 % ***
18.1 % ***
Threshold for statistical significance ns : p> 0.05, Not significant;* p : 0.01 to 0.05, Significant, ** p : 0.001 to 0.01, Very significant, ***p >0.001, strongly significant
Table 6 Effect of the active ingredient and placebo on a and b parameters.
26
SOFW-Journal | 140 | 4-2014
CO S MET IC S
I N F L A MMAT IO N
hand, heme oxygenase-1 (HMOX-1) is an
enzyme degrading heme into biliverdin,
carbon monoxide (CO), and free iron.
HMOX-1 protects endothelial cells from
apoptosis, is involved in blood-vessel relaxation regulating vascular tone, and
participates in blood-vessel formation
by means of angiogenesis and vasculogenesis (16). The regulation of vascular
tone is due to the relaxation of vascular
smooth muscle cells (17). HMOX-1 has
been also recognized to have major immunomodulatory and anti-inflammatory properties (17, 18), which have been
demonstrated in HMOX-1 knockout mice
and a human case of genetic HMOX-1
deficiency. Thus, the literature data have
shown that both VEGFR3 and HMOX-1
genes are involved in anti-inflammatory
pathways. Our study demonstrated that
the active ingredient is able to induce
the expression of the HMOX-1 gene,
therefore most probably activating
these anti-inflammatory properties.
Clinical study performed under the control of a dermatologist on human volunteers suffering of dark circles and bags
under eyes have shown the efficacy of
the active ingredient after 15 and 30
days of treatment. The improvement of
dark circles pigmentation was confirmed
by the significant variation of a and
b parameters measured: decrease of
a parameter by about -14 % to -18 %
and increase of b parameter by about
+11 % and +18 % respectively after 15
and 30 days of treatment.
The reduction of bags volumes due to
oedema under eyes by about -8 % and
-10 % after 15 and 30 days of treatment
has demonstrated the improvement of
lymphatic drainage provided by the active ingredient
Schematic representation showing the
anti-inflammatory action of the active
ingredient at different level of NF- B
pathway. (Fig. 8)
■ Conclusions
Dark circles and bags are reported to be
one of the biggest concerns for consumers all over the world and more precisely for Asiatic population (Mintel data),
right after wrinkles. Both dark circles and
under eyes puffiness are associated with
a lower self esteem and are reported to
have sometimes social impact, as people
believe that dark circles are associated
with a bad quality of life. Up to now,
the toolbox of formulators was limited
to few ingredients (caffeine, esculin) or
pigments to address this skin issue. By
combining three derivatives of gallic acid (gallic acid glucoside, epigallocatechin
Treatment
Concentration
Test compound
D0
D15
D30
Dark circles quotation
(mean±sem)
Dark circles quotation
(mean±sem)
Dark circles quotation
(mean±sem)
2.6±0.101
1.3±0.088
1.6±0.155
2.6±0.101
1.5±0.102
2±0.161
Placebo
Active Ingredient
glucoside and propyl gallate), which are
stabilized forms of gallic acid, it has been
possible to obtain significant improvement of dark circles and puffiness on
human volunteers in 15 to 30 days.
The suggested mechanism of action for
this active ingredient in these experiments was the inhibition of the NF- B
mediated inflammation. The inhibition
is due to the interaction of the active
ingredient with the inflammatory NF- B
pathway. Indeed, if the active ingredient was not able to block the nuclear
translocation of NF- B nuclear factor,
we have shown the strong effect of the
active ingredient to decrease the transcriptional activation of inflammatory
markers (IL 8, CXCL-1) known to be under
the control of NF- B nuclear factor after
its nuclear translocation.
This anti-inflammatory like activity was
also detected through the stimulation
of the transcription of two important
markers HMOX-1 and VEGFR3 known for
their anti-inflammatory properties. The
reduction of the volume of the bags is
expected to be mediated through the
activation of the VEGFR3 factor by the
active ingredient.
This study has highlighted a new efficient target »the NF- B inflammatory
pathway« to treat bags and dark circles
under eyes. The active ingredient shows
3%
Table 7 Clinical quotation of dark circles at D0, D15 and D30 by dermatologists.
Treatment
Test compound
Concentration
Placebo
Active
Ingredient
3%
D0
D15
D30
% of variation
volume
(mean±sem)
in mm3
volume
(mean±sem)
in mm3
volume
(mean±sem)
in mm3
D15 versus D0
D30 versus D0
21.7±1.334
20.4±1.308
20±1.344
(-6.2 %)**
(-8.1 %)**
22.1±1.503
20.2±1.336
19.8±1.340
(-8.4 %)***
(-10.1)***
Threshold for statistical significance ns : p> 0.05, Not significant;* p : 0.01 to 0.05, Significant, ** p : 0.001 to 0.01, Very significant, ***p <0.001, strongly significant
Table 8 Effect of the active ingredient and placebo on the volume of the bags under eyes.
28
SOFW-Journal | 140 | 4-2014
More Facts, Less Illusions
www.ifscc2015.com
CO S MET IC S
IN F L A MMAT IO N
a direct action on Nf- B pathway by decreasing the transcription rate of NF- B
mediated inflammatory markers such as
TNF alpha, IL1, IL8, CXCL1, and by reduc-
ing the release of CXCL1 and IL8 in the
tissue, which leads to the inhibition of
the inflammatory cascade. It also shows
an indirect interaction with the NF- B
signalling pathway by increasing the
transcription rate of HMOX-1 known to
block nuclear translocation NF- B factor. Meanwhile, the activation of the
transcription rate of VEGFR3 can explain
the action on the oedematous bags visible under eyes by the improvement of
draining properties of lymphatic vessels,
which are under the VEGFR3 control.
References
Fig. 8 Schematic representation of the inflammatory process and the mechanism
of action of the active ingredient in inflammatory condition.
30
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CO SMET ICS
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Abbreviations:
NHEK (Normal Human Epidermal Keratinocytes),
NF- B (Nuclear Factor-kappa B), TNF- (Tumor
Necrosis Factor alpha), IL1- (Interleukin-1 alpha),
IL8 (Interleukin-8), CXCL1 (Chemokine C-X-C motif ligand 1), VEGFR3 (Vascular endothelial growth
factor receptor 3), HMOX-1 (Heme Oxygenase -1).
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vascular bed: from molecular mechanisms to
therapeutic opportunities. Antioxid Redox
Signal, 2008. 10(10): p. 1767-812.
(17) Immenschuh, S. and H. Schroder, Heme oxygenase-1 and cardiovascular disease. Histol
Histopathol, 2006. 21(6): p. 679-85.
(18) Paine, A., et al., Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic
potential. Biochem Pharmacol, 2010. 80(12):
p. 1895-903.
*Authors´ address:
Dr. Hanane Chajra
Dr. Daniel Auriol
Dr. Kuno Schweikert
Dr. Fabrice Lefevre
Induchem AG
Industriestr. 8
8604 Volketswil
Switzerland
www.induchem.com
■
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(10) Massaro, F.C., et al., Cerumen of Australian stingless bees (Tetragonula carbonaria): gas chromatography-mass spectrometry fingerprints and
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