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Citrus Flavonoids with Skin Lightening Effects – Safety and Efficacy Studies

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Citrus Flavonoids with Skin Lightening Effects – Safety and Efficacy Studies

Abstract

A youthful appearance is not only related to the absence of wrinkles but also to skin radiance and an even skin tone. Many cosmetic ingredients are used to lighten skin by the inhibition of tyrosinase, the rate-limiting enzyme in melanin formation. Flavonoids, as major constituents of the human diet, have many well-known beneficial qualities. The aim of this study was to find the ideal composition of flavonoids from various citrus extracts for an effective skin lightening cosmetic ingredient. A chosen blend of citrus extracts was tested for safety and in vitro efficacy. No cytotoxic effects up to 0.8mg/ml could be detected and inhibition of human tyrosinase was 60% at 4mg/ml. The blend of citrus extracts was liposomal-encapsulated, incorporated into a cosmetic formulation at 1%, and then tested for stability and skin irritation capacity, as well as eye irritation and mutagenic potential and exhibited a very good safety profile. Finally the product was studied in vivo on Caucasian skin for efficacy during 56 days and showed marked skin brightening effects at a concentration of 1% (0.4mg/ml). This liposomal-encapsulated citrus flavonoid blend is effective at 1% in a cosmetic formulation to fade age spots and brighten skin tone.
12-2010
English Edition
International Journal for Applied Science
• Personal Care • Detergents • Specialties
S. Kiefer, M. Weibel,
J. Smits, M. Juch,
J. Tiedtke, N. Herbst:
Citrus Flavonoids with
Skin Lightening Effects –
Safety and Efficacy Studies
46 SOFW-Journal | 136 | 12-2010
COSMETICS
SKIN LIGHTENING
tion of tyrosine to dopaquinone (4). The
subsequent steps of melanin formation
from dopaquinone can occur sponta-
neously at physiological pH. Dopaquinone
is oxidized to dopachrome and through
further oxidation reacts to melanin.
Many of the currently used depigment-
ing agents are tyrosinase inhibitors, as
it seems the most straightforward way
to
inhibit melanin formation. Although
more
recent in-depth knowledge of
melanocyte biology and the processes
underlying melanin formation has pro-
duced other interesting targets for the
treatment of hyper-pigmentation, such
as the inhibition of translation and matu-
ration of tyrosinase, stimulation of degra-
dation
and acceleration of skin turnover.
The best known skin whitening agent is
hydroquinone, which has been used for
over 50 years now although some con-
troversy about it has come up in recent
years. Long term use of high concentra-
tions of hydroquinone can produce side
effects like ochronosis, a thickening and
darkening of skin, especially of dark-
skinned individuals (5) and general safe-
ty concerns have been brought up by
regulatory agencies all over the world.
Therefore there is still a need for safe and
effective skin lightening products and
the hunt for natural skin lighteners (6) is
ongoing.
Flavonoids form major constituents of
the human diet as they contribute to the
flavour and colour of many fruits and
vegetables. Their beneficial antioxidant
effects are thoroughly studied and es-
tablished.
The highest content of citrus flavonoids
can be found in unripe citrus fruits (7).
S. Kiefer, M. Weibel, J. Smits, M. Juch, J. Tiedtke, N. Herbst*
Citrus Flavonoids with Skin Lightening Effects –
Safety and Efficacy Studies
Introduction
With ageing of the skin pigmentary dis-
orders such as age spots, melasma, and
uneven skin tone become more frequent.
An even, bright and unblemished skin is
one of the main criteria for a young
looking appearance (1-3). Hence skin
lightening products form a major seg-
ment in the cosmetic industry and are a
part of many anti-ageing products that
reach the market every year.
Melanin, the substance responsible for
the pigmentation of the skin, is produced
to protect the DNA from harmful, muta-
genic UV radiation. Age spots or solar
lentiges are age-related and UV irradia-
tion-induced pigmented spots that usu-
ally occur on sunlight-exposed areas
such as the face, the back of the hands,
and the upper chest.
The rate-limiting enzyme in melanin for-
mation is polyphenol oxidase tyrosinase
(EC 1.14.18.1) which catalyses the oxida-
Ayouthful appearance is not only related to the absence of wrinkles
but also to skin radiance and an even skin tone. Many cosmetic in-
gredients are used to lighten skin by the inhibition of tyrosinase, the
rate-limiting enzyme in melanin formation. Flavonoids, as major con-
stituents of the human diet, have many well-known beneficial qualities.
The aim of this study was to find the ideal composition of flavonoids from
various citrus extracts for an effective skin lightening cosmetic ingredient.
A chosen blend of citrus extracts was tested for safety and
in vitro
efficacy.
No cytotoxic effects up to 0.8mg/ml could be detected and inhibition of
human tyrosinase was 60% at 0.4mg/ml. The blend of citrus extracts was
liposomal-encapsulated, incorporated into a cosmetic formulation at 1%,
and then tested for stability and skin irritation capacity, as well as eye irri-
tation and mutagenic potential and exhibited a very good safety profile.
Finally the product was studied
in vivo
on Caucasian skin for efficacy dur-
ing 56 days and showed marked skin brightening effects at a concentration
of 1% (0.4mg/ml).
This liposomal-encapsulated citrus flavonoid blend is effective at 1% in a
cosmetic formulation to fade age spots and brighten skin tone.
Abstract
48 SOFW-Journal | 136 | 12-2010
COSMETICS
SKIN LIGHTENING
These unripe fruits can be collected by
thinning out, which is necessary for a
good harvest of ripe fruits later on (8).
Whole ground fruits are extracted with
a water methanol mixture to yield the
highest flavonoid content.
The aim of this study was to find the ide-
al composition of flavonoids from vari-
ous citrus fruit extracts to use as an ef-
fective skin lightening cosmetic ingredi-
ent.
Materials and Methods
All solvents from Merck, all other chemi-
cals from Sigma Aldrich
Raw material
The various citrus extracts
were bought
from different extract
manufacturers
and analyzed by HPLC for their content
of naringin, narirutin, hesperidin, and
neohesperidin.
HPLC instrumentation
HPLC separation was carried out on a
Surveyor HPLC system with degasser, bi-
nary high pressure mixing pump, column
thermostat, and
auto sampler. The HPLC
was coupled to a DAD detector and da-
ta acquisition and processing was per-
formed using
ChromQuest Chromatog-
raphy Data System (CDS) (all Thermo
Fisher Scientific).
Separation of flavonoids
On a C18 column (Utisphere OBD, 120Å,
150 x 4.6 mm, 5µm) an isocratic elution
with 75% water, 10% methanol, 10%
acetonitrile, and 5% acetic acid 99%
was performed with a flow of 1 ml/min
and subsequent UV detection at 285 nm.
Tyrosinase inhibition
Mushroom tyrosinase (333 U/ml, EC
1.14.18.1, Sigma Aldrich, Switzerland)
was incubated in 18 mM phosphate
buffer, extracts and tyrosine solution (fi-
nal concentration of 1 mM) were added
and absorbance was measured over 5
hours at 490 nm.
Cellular human tyrosinase activity test
Normal human epidermal melanocytes
(NHEM, Bioalternatives, France) were in-
cubated for 72 hours with test compound
and control. After incubation, culture
medium was removed and cells washed
with PBS. The tyrosinase enzyme was ex-
tracted with Triton X in PBS and then in-
cubated with 2 mM L-3,4-dihydroxyphe-
nylalanine (L-DOPA) as substrate. After
incubation enzymatic activity was mea-
sured at 540 nm (ThermoMax micro plate
reader, Molecular Devices).
Cytotoxicity
Normal Human Epidermal Melanocytes
(NHEM, Bioalternatives, France) were
incubated with concentrations from
0.0015 to 1.14 mg/ml of flavonoid mix-
ture for 72 hours. Then a solution of
0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT)
was added, after incubation at 37 ° C for
4 hours, the media was discarded and
100 ul DMSO was added to each well. The
optical density of the dissolved residues
was measured at 540 nm (Microplate
reader, Molecular devices).
Liposomal encapsulation
The flavonoid mixture was mixed with
glycerin, lecithin, tocopherol, sodium
ascorbate,
and homogenized with a high
pressure homogenizer (Microfluidics,
Lampertheim, Germany). Subsequently
the liposome size was determined using
a Zetasizer Nano ZS90S from Malvern In-
struments (Worcestershire, United King-
dom).
Formulation
1% of the liposomal solution was incor-
porated into a lotion (Table 1) for appli-
cation studies (patch tests and in vivo ef-
ficacy). The formulation was tested for
stability towards temperature and time.
Patch tests
For both, single and repeated patch tests
25 healthy volunteers between 18 and
60 years were selected. The dorsal skin
was cleaned with 70% alcohol before
Component INCI Weight (g) Function
Water Aqua 78.6 Solvent
NaOH 10% Aqua, Sodium Hydroxide 0.5 pH-adjustment
Keltrol CG SFT Xanthan Gum 0.4 Emulsion stabilizer
Crodafos CES Cetearyl Alcohol, Dicethyl Phosphate, Ceteth-10 Phosphate 3.0 Emulsifier
Myritol 318 Caprylic/Capric Triglyceride 13.0 Skin-conditioning agent
Preservative Phenoxyethanol, Ethylhexylglycerin q.s. Preservative
Silicone Oil Dimethicone 0.5 Antifoaming agent
Shea Butter Butyrospermum Parkii (Shea Butter) 3.0 Skin-conditioning agent
Flavonoid mixture, Glycerin, Aqua, Lecithin, Citrus Paradisi (Grapefruit) Fruit Extract, 1.0 Active
Citrolumine 8™ Citrus Aurantium Amara (Bitter Orange) Fruit Extract, Sodium
Ascorbate, Tocopherol, Heliantus Annus (Sunflower) Seed Oil
Total 100.0
Table 1 Frame formulation for the liposomal encapsulated citrus flavonoid blend, as used in the application studies
50 SOFW-Journal | 136 | 12-2010
COSMETICS
SKIN LIGHTENING
product application and then 1% of li-
posomal-encapsulated flavonoid mix-
ture in a lotion was applied using a Finn
Chamber (7 mm). The product was left in
contact with the skin for 48 hours (sin-
gle patch test) or 24 hours and reapplied
4 times (repeated patch test). The cuta-
neous reactions were evaluated 15 min-
utes, 1 hour, and 24 hours after removal
of the product. One chamber with dis-
tilled water was used as negative control
and for the single patch test only a pos-
itive control was applied with 0.5% sodi-
um dodecyl sulphate (SDS).
Application study
Caucasian skin: Six healthy female vol-
unteers of Caucasian skin type (all Fitz-
patrick skin types II) were recruited for
application of the lotion or a control
(formulation without active ingredient).
The cream was applied twice a day dur-
ing 8 weeks on the back of the hand and
the face. Melanin Index was measured on
the back of the hand and the face with
a Skin Pigment Analyzer, SPA99 (Courage
&Khazaka, Köln, Germany) and pho-
tographs were taken from the face and
back of the hand with VisioFace Quick
(Courage &Khazaka).
Asian skin: Six healthy female volun-
teers of Asian skin type were recruited
for application of the lotion on the ex-
ternal forearm. The lotion was applied
twice daily during 8 weeks. Skin color
was measured with a Chromameter CR300
(Konica Minolta, Dietikon, Switzerland).
All volunteers were measured and pho-
tographed 3 times; at day 0 (baseline),
day 28, and day 56.
Results
Raw material
The various citrus extracts were tested by
chromatographic separation for their
content of narirutin, naringin, hesperidin,
and neohesperidin.
Two extracts were chosen, one from un-
ripe citrus paradisi (grapefruit) and one
from unripe citrus aurantium amarum
(bitter orange) fruit and mixed. The re-
sulting mixture of raw materials result-
ed in the following flavonoid content
of 4.9% narirutin, 22.0% naringin, 1.0%
hesperidin, and 5.3% neohesperidin (Fig. 1).
Tyrosinase inhibition
(mushroom tyrosinase)
Inhibition of mushroom tyrosinase was
tested in a concentration range from
0.025 to 3 mg/ml. Up to 1 mg/ml inhibi-
tion increased dose dependently up to
40% of untreated control, in concentra-
tion higher than 1 mg/ml there was no
further increase possible (Fig. 2). The
flavonoid mixture showed an estimated
IC50 of 0.75mg/ml.
Cellular human Tyrosinase activity test
Tyrosinase activity was tested after incu-
bation of NHEM with 0.004 to 0.4 mg/ml
flavonoid mixture for 72 hours and sub-
sequent tyrosinase extraction of the
cells. As a control 0.02 mg/ml of kojic acid
was used. A concentration of 0.004 mg/
ml of flavonoid mixture reduced the ty-
rosinase activity to 66%, 0.04 mg/ml to
65%, and 0.4 mg/ml to 40%, whereas ko-
jic acid reduced tyrosinase activity to
61% at 0.02mg/ml (Fig. 3).
Viability/ Cytotoxicity
Up to 0.4 mg/ml flavonoid mixture via-
bility was increased up to 130%, at
1.12 mg/ml a reduction of viability to
83% was measured (Fig. 4). A concen-
tration of 3.3 mg/ml resulted in no re-
maining viability (data not visible).
-
F
ig. 1
HPLC chromatogram of the flavonoid mixture
-
F
ig. 2
Mushroom tyrosinase inhibition of flavonoid mixture
SOFW-Journal | 136 | 12-2010 51
COSMETICS
SKIN LIGHTENING
Safety tests
Ames reverse mutation assay (OECD No.
471) using Salmonella typhimurium and
Escherichia coli showed no mutagenic
potential of the liposomal-encapsulated
flavonoid mixture.
The bovine corneal opacity and perme-
ability assay (BCOP, OECD No. 437) of the
liposomal-encapsulated flavonoid mix-
ture revealed no eye irritation potential
of the product.
Liposomal encapsulation
Liposomes showed an average particle
size of 120 nm which was stable over
several production batches. They formed
a transparent, stable solution and no
preservation was necessary to keep the
solution from microbiological conta -
mination. A Pharmacopoeia Europea 6
preservation test for topical products
was performed and received a grade B
evaluation.
Stability of frame formulation
The formulation for the application
study was stable for 6 month at room
temperature, at 40°C, and at 2-8°C in the
refrigerator.
Patch tests
The single and repeated human patch
tests showed no irritation and sensitiza-
tion potential of the liposomal-encapsu-
lated flavonoid mixture in a lotion at 1%,
which corresponds to 0.4 mg/ml of
flavonoid mixture.
Application Study
Caucasian Skin
The application study on Caucasian skin
(n=6) showed skin lightening effects and
age spots brightening after 28 and 56
days of application compared to base-
line. The effects on age spot brightening
were slightly more apparent than the
lightening effects on skin tone. Both on
hand (Fig. 5) and face (Fig. 6) the re-
sulting birightening effects were in the
same range. Fig. 7 shows the spot-fad-
ing and skin brightening effects of a
VisoFace Quick picture of the back of the
hand on day 0 and day 56.
-
F
ig. 3
Tyrosinase inhibition of cellular tyrosinase extracted from NHEM compared
to untreated control
-
Fig. 4 MTT viability assay with flavonoid mixture from 0.0015 mg/ml up to 1.116 mg/
ml
-
Fig. 5
Skin brightening effect of liposomal-encapsulated flavonoid mixture at 1%
in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied on the back of
the hand twice daily for 28 and 56 days compared to baseline (day 0)
52 SOFW-Journal | 136 | 12-2010
COSMETICS
SKIN LIGHTENING
Asian Skin
After promising results on Caucasian
skin an additional study on the external
forearm of Asian volunteers (n=3) was
performed and showed a lightening ef-
fect compared to baseline (day 0), which
was significantly present after 56 days
(Fig. 8).
Discussion
The flavonoid mixture was effective in
vitro. Good inhibtion has been shown in
the mushroom tyrosinase enzyme inhi-
bition test with an estimated IC50 of
about 0.75 mg/ml.
The cellular human tyrosinase inhibition
assay resulted in stronger tyrosinase in-
hibition, there the estimated IC50 was
0.24 mg/ml of flavonoid mixture. Origi-
nally the potential lighthening effect of
citrus flavonoids was discovered by mush-
room tyrosinase inhibition assay (9).
A range of tests showed that the citrus
flavonoid blend is safe for application on
skin. No cytotoxicity has been observed
in concentrations that were applied. In
the single and repeated patch tests no ir-
ritation or sensitisation potential was
found and also no mutagenic potential
(as tested by Ames Test, data not shown)
and no eye irritation was detectable
(BCOP test, data not shown). These find-
ings show the excellent safety profile of
the flavonoid blend for use as a cosmet-
ic ingredient.
In vivo studies showed that it was very
effective in brightening the skin tone and
had an even stronger effect on the fad-
ing of age-spots, resulting in a more even
skin tone. To get more accurate data with
lower standard deviations a bigger study
with at least 20 volunteers would be nec-
essary. The high standard deviation was
due to the small test groups (n=6 for
Caucasian and n=3 for Asian Skin). After
28 days effects were already visible and
were enhanced after 56 days, thus im-
plying that stronger brightening effects
can be obtained after longer application.
These data suggest that the liposomal-
encapsulated citrus flavonoid blend
(Citrolumine 8™ from Cosmetochem AG,
Switzerland) can be used at 1% in a cos-
metic lotion as a very safe and effective
skin lightening ingredient.
-
Fig. 6
Skin brightening effect of liposomal-encapsulated flavonoid mixture at 1%
in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied to the face twice
daily after 28 and 56 days compared to baseline (day 0)
-
Fig. 7
Skin brightening and spot-fading effect of liposomal-encapsulated flavonoid
mixture at 1% in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied
to the back of the hand twice daily for 56 days. Left picture baseline at day 0, right
picture after twice daily application during 56 days
Day 0 Day 56
-
Fig. 8
Skin brightening effect of liposomal-encapsulated flavonoid mixture at 1%
in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied to the external
forearm of asian volunteers twice daily after 28 and 56 days compared to baseline
(day 0). After 56 days a sigificant brightening effect can be measured
54 SOFW-Journal | 136 | 12-2010
Acknowledgements
These investigations were realized and
funded by Cosmetochem International
AG, Switzerland.
The authors’ would like to thank Dr. Ru-
di Wajda from Lipoid GmbH, Ludwigs-
hafen, for liposomal encapsulation and
measurements and Daniel Lisibach from
Cosmetochems analytical lab for all the
HPLC analyses.
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* Authors’ address:
Sabine Kiefer, Michaela Weibel
Julian Smits, Mathias Juch
Jane Tiedtke, Norbert Herbst
Cosmetochem International AG
Sennweidstrasse 44/46
6312 Steinhausen
Switzerland
Email: sabine.kiefer@cosmetochem.ch
SKIN LIGHTENING
COSMETICS
... Taken together, these studies demonstrate that ↑ -glucocerebrosidase activity. [33] Evidence-Based Complementary and Alternative Medicine 5 [34] Keratinocytes were treated with hesperidin (220 g/ml) for 24 hr, followed by UVA irradiation (10 J/cm 2 [52] Evidence-Based Complementary and Alternative Medicine 7 Anti-oxidative stress [60] Evidence-Based Complementary and Alternative Medicine 9 Guinea pigs Hesperidin (40mg/kg) was orally given 1 hr prior to injection of carrageenan. ...
... In contrast, other studies demonstrated that hesperidin did not affect melanin production in B16F10 murine melanoma cells [47][48][49]. However, most of other studies showed that both citrus extract and hesperidin inhibited melanogenesis in both murine B16-F10 melanoma cells and human melanocytes [36,44,[50][51][52][53]. For example, treatments with 50 M hesperidin for 48-72 hr induced 60% reduction in melanin content in murine B16-F10 melanoma cells and ≈30% reduction in human melanocytes [50]. ...
... Upregulation of expression levels of tyrosinase, TRPs, and MITF can increase melanin production [117,118]. A number of studies demonstrated that hesperidin decreased expression levels and activity of tyrosinase, TRPs, and MITF in both B16 mouse melanoma cells and human melanocytes [36,[50][51][52]. Moreover, hesperidin could activate adrenergic receptor, leading to induction of aggregation of melanophores in B. melanostictus, suggesting that hesperidin-induced skin lightening is mediated by adrenergic receptor [119]. ...
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... The result is similar to the paper by Kiefer et al. that shows skin melanin index was reduced by snake fruit ethanolic extract. Considering that the study concluded that flavonoid from various citrus extracts was very effective in brightening the skin tone, it means that flavonoid from snake fruit ethanolic extracts might play a role in decreasing human skin melanin index 12 (Table 1 and 2). ...
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Introduction: The whitening skin product market has been growing significantly in Asia Pacific. Indonesia is one of those countries that mainly think that having a white color skin is simply beautiful. Therefore, it motivates Indonesian women to buy more whitening product for their beauty needs. Objective: The purpose of this research is to find raw material for whitening product from Indonesian plants that can be useful as skin lightening agents. This study investigated the potential of snake fruit (Salacca edulis Reinw var. Bongkok) ethanolic extract as skin lightening agent. Fruits of Salacca edulis Reinw contains flavonoids which have been reported to play a part in skin depigmentation. Methods: A randomized double-blind study on 17 human volunteers showed marked skin lightening effects using cream containing 3% extract as there was a significant reduction in skin melanin index which compared favorably with the base cream (p <0,05). Results: The result strongly suggested that snake fruit ethanolic extract can be used as raw material for skin lightening agents and therefore Indonesia dependency on imported raw materials could be reduced.
... The result is similar to the paper by Kiefer et al. that shows skin melanin index was reduced by snake fruit ethanolic extract. Considering that the study concluded that flavonoid from various citrus extracts was very effective in brightening the skin tone, it means that flavonoid from snake fruit ethanolic extracts might play a role in decreasing human skin melanin index 12 (Table 1 and 2). ...
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Introduction: The whitening skin product market has been growing significantly in Asia Pacific. Indonesia is one of those countries that mainly think that having a white color skin is simply beautiful. Therefore, it motivates Indonesian women to buy more whitening product for their beauty needs. Objective: The purpose of this research is to find raw material for whitening product from Indonesian plants that can be useful as skin lightening agents. This study investigated the potential of snake fruit (Salacca edulis Reinw var. Bongkok) ethanolic extract as skin lightening agent. Fruits of Salacca edulis Reinw contains flavonoids which have been reported to play a part in skin depigmentation. Methods: A randomized double-blind study on 17 human volunteers showed marked skin lightening effects using cream containing 3% extract as there was a significant reduction in skin melanin index which compared favorably with the base cream (p <0,05). Results: The result strongly suggested that snake fruit ethanolic extract can be used as raw material for skin lightening agents and therefore Indonesia dependency on imported raw materials could be reduced.
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Evolutionary psychology suggests that skin signals aspects of mate value, yet only limited empirical evidence exists for this assertion. We sought to study the relationship between perception of skin condition and homogeneity of color/chromophore distribution. Cropped skin cheek images from 170 girls and women (11-76 years) were blind-rated for attractiveness, healthiness, youthfulness, and biological age by 353 participants. These skin images and corresponding melanin/hemoglobin concentration maps were analyzed objectively for homogeneity. Homogeneity of unprocessed images correlated positively with perceived attractiveness, healthiness, and youthfulness (all r > 0.40; P < .001), but negatively with estimated age (r = -0.45; P < .001). Homogeneity of hemoglobin and melanin maps was positively correlated with that of unprocessed images (r = 0.92, 0.68; P < .001) and negatively correlated with estimated age (r = -0.32, -0.38; P < .001). Female skin only was studied. Skin color homogeneity, driven by melanin and hemoglobin distribution, influences perception of age, attractiveness, health, and youth.
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According to evolutionary psychology, the preference for some facial characteristics reflects adaptations for mate choice because they signal aspects of mate quality. Although morphological features such as facial symmetry and sexually dimorphic traits have been studied extensively in recent years, little is known about skin condition in this context. The preferences for young and healthy looking skin could offer an explanation as to why women place such an importance on the condition of their skin and its refinement through e.g., cosmetic products. Recent research showed that facial skin colour distribution significantly influences the perception of age and attractiveness of female faces, independent of skin surface topography cues. However, the relative effect of skin colour distribution and topography cues on age and health perception remains to be investigated. We present data showing that both skin colour distribution and skin surface topography cues not only significantly influence the perception of female facial age and health but also convey differential information with regard to the strength of these effects. Our data indicate that skin surface topography cues account for a large proportion of variation in facial age perception, whereas skin colour distribution seems to be a stronger health cue.
Why some women look young for their age Color homogeneity and visual perception of age, health, and attractiveness of female facial skin
  • Da Gunn
  • H Rexbye
  • Ce Griffiths
  • Pg Murray
  • A Fereday
  • Sd Catt
  • Cc Tomlin
  • Bh Strongitharm
  • Di Perrett
  • M Catt
  • Ae Mayes
  • Ag Messenger
  • Mr Green
  • F Van Der Ouderaa
  • Jw Vaupel
  • K Christensen
  • Pj Matts
  • B Fink
  • K Grammer
  • M Burquest
Gunn DA, Rexbye H, Griffiths CE, Murray PG, Fereday A, Catt SD, Tomlin CC, Strongitharm BH, Perrett DI, Catt M, Mayes AE, Messenger AG, Green MR, van der Ouderaa F, Vaupel JW, Christensen K. Why some women look young for their age. PLoS One 2009;4 (12):e8021 (3) Matts PJ, Fink B, Grammer K, Burquest M. Color homogeneity and visual perception of age, health, and attractiveness of female facial skin. J Am Acad Dermatol 2007;57 (6):977-84