Skin-Lightening Agent with Different Pathways of Action ... - Dermage

COSMETICS
SKIN-LIGHTENING
S. John, P. Lorenz, R.-D. Petersen, M. Heldermann, S. Borchert
Skin-Lightening Agent with Different
Pathways of Action on Melanogenesis
Keywords: Bellis perennis, skin whitening, tyrosinase expression, tyrosinase inhibition, ROS,
superoxide (O 2 .- ) scavening, melanin inhibition, α-MSH, endothelin-1 (ET-1),
MC1-R, melanosome uptake
Abstract
Anovel botanical ingredient - trade name: Belides TM - obtained from
daisy flower blossoms (Bellis perennis L.) consists of bioactive constituents
such as saponins (triterpene glycosides), polyphenols and
polysaccharides. The presence of these constituents suggests
that this active produces a variety of effects
which are also used in phytomedicine. Surprisingly,
Belides was found to exhibit a strong inhibitory effect
on melanogenesis (patent pending). The fraction of
polyphenols is known to inhibit tyrosinase enzymatic
activity. Results of a comparative study conducted with
Belides and arbutin, used at equivalent polyphenol concentrations,
revealed that Belides was about twice as active as
arbutin. Tests carried out on melanoma cells (B16V) have shown that Belides
controls transcription of tyrosinase expression, thus inhibiting its synthesis.
In addition, Belides has been found to significantly decrease the release
of the peptide hormone endothelin (ET-1), the binding capacity of alpha-MSH
(melanocyte-stimulating hormone) on the melanocortin receptor-
1 (MC1-R) and also melanosome uptake mechanisms. Furthermore,
results of a pilot study performed on human volunteers demonstrate the
in vivo skin-lightening efficacy of Belides. Belides is recommended for use
in skin-lightening cosmetics and in case of pigmentation disorders, hyperpigmentation
or age spots.
� Introduction
An even skin tone is an ideal of beauty
which seems to be reachable by using
special skin-lightening products supplied
by the cosmetic industry.
Cosmetic actives producing a skin-lightening
effect are expected to act in three
main directions:
1) Reaching a matching, consistent tone
of unevenly pigmented skin (difference
between covered and non-covered
skin parts).
2) Influence on pigmentation disorders
including freckles, age or sun spots,
melasma, acne spots from healed acne
eruptions etc.
3) Reduction of natural skin pigmentation
to give appearance of a lighter
skin tone (mainly in Asian countries).
To achieve any of these three targets – all
of them aiming to influence melanogenesis
– different mechanisms will be
involved either individually or in combination
(synergistic effect). Depigmentation
can be accomplished by specific interaction
of an active (Fig. 1):
• Before melanin synthesis
• During melanin synthesis
• After melanin synthesis
The most effective and rapid form of skin
lightening consists in blocking induction
of melanin synthesis. The UVB fraction of
sunlight triggers keratinocytes to release
proinflammatory mediators such as the
melanogen endothelin-1 (ET-1). ET-1 is a
protein which acts on melanocytes via a
receptor-mediated signal transduction
pathway. In a paracrine fashion it stimulates
melanocyte proliferation, migration,
dendrite formation and melanogenesis
by transcriptional control of tyrosinase
expression (2,3). Either inhibition
of ET-1 expression or antagonistic action
on the ET-1 receptor inhibit melanogenesis
before the latter starts.
Another triggering factor of melanogenesis
is involved in upstream control
of melanin synthesis: the melanocytestimulating
hormone α-MSH (4). Actives
acting as antagonists of this mediator
are able to block the α-MSH receptor
MC-1R.
A common way of down-regulating
melanogenesis during synthesis consists
in reducing the enzyme activity of ty-
40 SÖFW-Journal | 131 | 7-2005

Belides lightens
and evens skin
tone.
Belides is produced by
gentle extraction of carefully
collected flowers of Bellis
perennis. The extract obtained
shows skin lightening activity.
makes the skin look like ivory Belides
CLR‘s new skin care active Belides is designed for use in depigmenting formulations. Commonly used skin lightening
actives inhibit melanogenesis by reducing tyrosinase activity. Belides does more than that. Due to modulation of other
biochemical pathways involved in melanin synthesis it produces synergistic effects (patent pending) by: Inhibition of
tyrosinase, transcriptional control of tyrosinase expression and reduction of the pro-melanogenic mediator endothelin.

COSMETICS
SKIN-LIGHTENING
rosinase (1). Tyrosinase catalyzes two oxidative
steps in melanin synthesis. For
this reason, antioxidants like polyphenols
or scavengers of reactive oxygen
species (ROS) are also able to interfere
with melanin synthesis.
Finally, depigmentation can be induced
after melanin synthesis has finished
by inhibiting the transfer of mature
melanosomes to keratinocytes (5). Several
studies have focussed on the interaction
between melanocytes and keratinocytes
during the transfer process
(6). The most common way to achieve
lightening of naturally pigmented skin is
to reduce efficiency of melanin synthesis,
e.g. by using tyrosinase inhibitors, antioxidants
and ROS-scavengers.
These different target fields of application
illustrate the advantage of a whitening
active which produces a cumulative
effect by acting on different pigmentation
pathways.
It will be reported on in vitro and in vivo
test results obtained with a new skinlightening
product Belides, demonstrating
the efficacy of skin-lightening activity
and the mechanisms of action.
The plant
Bellis perennis L., the so-called ‘daisy
flower‘ (Fig. 2), is a small perennial flowering
plant (between 2 – 10 cm tall), belonging
to the Asteraceae (= Compositae)
family. The plant is widely distributed
in Northern Europe, from the Atlantic
coast of Portugal and Spain to the
Western slope of the Kaukasus mountain,
including the British Isles and Southern
Scandinavia (7). The plant often grows on
pastures, meadows or grassland, were it is
collected.
Fig. 2 Daisy flower blossoms (Bellis perennis L.)
Fig. 1 Schematic Illustration of the possible approaches to interfere with melanogenesis
pathways (after Briganti et al. (1))
Bellis perennis has been used since ancient
time in traditional folk medicine
for treatment of dermatopathies like furunculosis,
suppuration and slow-healing
eczema. Furthermore, it has been applied
against cough, fever, inflammation,
dysmenorrhoe, amenorrhoe, headaches,
dizziness and sleeplessness (insomnia), as
well as in homeopathy (7). Phytochemical
investigations of Bellis perennis have
shown a wide spectrum of natural constituents
such as saponins (8), flavonoids
and other poly-phenolic compounds (7).
Here it is reported for the first time that
an active obtained from Bellis perennis
inhibits the melanogenesis in human
skin.
� Materials and Methods
Manufacturing process of Belides. Daisy
flower blossoms (Bellis perennis L.) were
gently extracted using an aqueous buffer
system and following an internal procedure.
Then, the extract was fractionated
by means of ultrafiltration.
Total polyphenol content of Belides was
measured after a modified literature
method using the Folin-Ciocalteu reaction.
Gallic acid was used as an external
standard for quantification.
Superoxide scavenging (O 2 .- ) capacity
of Belides was determined according to
a literature method decribed by Lorenz
et al. (10) using an enzymatic O 2 .- generation
(hypoxanthin-xanthinoxidase reaction)
that was visualized with NBT (nitroblue
tetrazolium, Sigma) yielding a
blue formazan product.
Tyrosinase enzymatic activity was measured
according to a modified literature
method (11). Test samples of Belides (0.5,
1.25, 2.5 and 10.0%) were incubated
with L-tyrosine (186.9 µM) and mushroom
tyrosinase (Sigma; 151.06 units/ ml)
for 5 min at 40°C and L-DOPAchrome formation
determined spectrophotometrically
at λ max = 490 nm.
Melanin inhibition assay. Mouse melanoma
cells (B16V) were seeded at 2 x
10 5 cells/ml in T 25 flasks (TPP) and preincubated
for 3 days in vitro (DIV) in RPMI
1640 medium (Biochrom; F1215) supplemented
with 10% fetal calf serum
(FCS), 2 mM L-glutamine and gentamycin
(50 mg/l) at 37°C in a humidified 5%
CO 2 atmosphere. Afterwards the medium
was removed and the Belides-containing
medium applied on the cell cultures
as well as on controls for 7 DIV. Cells
were trypsinated and lyzed with 2 ml tri-
42 SÖFW-Journal | 131 | 7-2005

chloroacetic acid (5%). After centrifugation
the precipitates were resuspended
in 1.3 ml sodium hydroxide (1 N).
The melanin absorption was measured at
475 nm against blank samples. Melanin
values were normalized on cell density.
Optical density of control samples was
set to 100%.
Enzymatic quantification of tyrosinase
expression. B16V mouse melanoma cells
were cultivated and treated as described
in the melanin inhibition assay method.
After trypsination the cells were lyzed
with 1.0% Triton X-100 (Sigma-Aldrich
Co.) in PBS and mixed with a L-DOPA solution
(0.1% L-DOPA in 0.1 M PBS, pH
6.0). The transformation of L-DOPA to
L-DOPAchrome was measurable within
15 - 30 min by the absorbance at 450 nm
and referenced at 630 nm by a microplate
reader (MRX; Dynex Technologies,
Inc.).
Endothelin-1 (ET-1) assay. ET-1 was quantified
by using a chemiluminescent immunoassay
(QuantiGlow; R&D Systems).
Human epidermal keratinocytes (HaCaT,
a gift of Prof. Fusenig; DKFZ Heidelberg;
Germany) were grown in Dulbecco’s Modified
Eagle Medium (DMEM), supplemented
with 5% FCS, 200 mM L-glutamine
and 50 mg/l gentamycin (all from Biochrom
KG, Berlin). Cells were seeded on
a 96-well MTP with 4.5 x 10 4 cells/well
and incubated for 48 h and further 3 DIV
with Belides-containing medium. Then
cells were stimulated for 24 h with 40
ng/ml Interleukin-1 (IL-1; Sigma-Aldrich
Co.). Afterwards the cells were used for
the QuantiGlow ET-1 ELISA. The resulting
luminescence reaction was monitored by
a luminescence reader (Fluoroscan Ascent
FL; Labsystems Ltd.; lag time: 1 min, reading
time: 0.5 sec).
The α-MSH assay. Mouse melanoma cells
(B16V) were seeded at 4.5 x 10 4 cells/well
in microtiter plates (MTP) and cultivated
in RPMI 1640 medium with 10% FCS for
2 DIV, followed by a sample application
for 72 h. Then fresh medium containing
1µM alpha-MSH-biotin (Phoenix Europa,
Karlsruhe) was applied for 2 h. After
removing unbound α-MSH, a streptavidin-horseradish
peroxidase (HRP) solution
(Sigma-Aldrich Co.) was added to
the cells. Then o-phenylendiamine-substrate
solution (Sigma-Aldrich Co.) was
applied and the resulting OD values were
read at 450 nm by MRX. Cells without
α-MSH-biotin served as a blank.
Phagocytosis activity of keratinocytes
as a function of melanosome uptake
HaCaT cells were seeded at 1 x 10 3 cells/
well in DMEM for 48 h in a MTP. Belidescontaining
medium was applied for further
3 DIV, followed by irradiation of the
plates with 0.3J/cm 2 UVA and 0.03J/m 2
UVB with the UV lamp SOL 500 (Dr. Hönle;
Munich, Germany). Immediately after
irradiation a FluoSpheres solution (beads
size 1.0 µm, Molecular Probes) was added
for 24 h. Then the cells were lyzed with
1.0% Nonident P40, 0.01% sodium dodecyl
sulfate in 0.1 M Tris-HCL (pH 7.2).
The fluorescence emission of the lysates
was detected by a fluorescence reader
(Fluoroscan Ascent FL; Labsystems Ltd.)
at an excitation/emission wavelength of
585 nm/612 nm. Cells not treated with
FluoSpheres served as a blank.
Skin-lightening efficacy test on human
volunteers (in vivo pilot study). The in
vivo pilot study was performed as a double
blind study by Derma Consult GmbH
(Alfter, Germany). The trials were carried
out by using the inner side of the forearm
as test side. After the first measurement
(Chromameter), the test products
were applied twice daily. Measurements
were evaluated during treatment on day
14, 28 and 35 about four hours after the
last daily application. The L-values reflected
the skin color were measured
with a Minolta Chromameter CR 300
(Minolta, Japan) in compliance with the
Commission International de l’eclairage
Scheme 1 Biosynthesis of melanin
COSMETICS
SKIN-LIGHTENING
(CIE) system. Each L-value has an average
of three recordings. Measurements
are according to the guidelines of the
European Society of Contact Dermatitis
(12). Probability values (p) less than 0.05
were considered to be significant.
� Results and Discussion
In the recent years there has been a growing
demand for cosmetic skin-lightening
products. Whereas in Western countries
these products are mostly used for the
prevention or treatment of irregular (hyper)pigmentation,
like freckles, age- or
sunspots, in Asia and Africa skin-lightening
formulations are also applied to
make the skin lighter and brighter.
Conventional compositions developed
for depigmentation generally contain
ingredients that have antioxidant and
tyrosinase-inhibiting activity (e.g. ascorbic
acid, kojic acid, hydroquinone derivatives
like arbutin etc.; for review see:
(1)).
In the search for a new potential skinlightening
product with a broader spectrum
of action on melanogenic pathways
we are focusing here on Belides, a
new active which is produced from daisy
flowers (Bellis perennis).
Melanin, responsible for pigmentation of
the skin, is produced in melanosomes
which are located in the melanocytes.
The synthesis of melanin involves oxidative
processes, whereby L-tyrosine is
converted into L-dihydroxy phenylalanine
(L-DOPA) by the enzyme tyrosinase.
L-DOPA is then finally converted
to melanin by a complex chain of oxidative
reactions (Scheme 1).
Transcription and expression of the ty-
SÖFW-Journal | 131 | 7-2005 43

COSMETICS
SKIN-LIGHTENING
rosinase enzymatic protein is an upstream
process before melanin formation
can start (Fig. 1). By inhibiting transcription
of RNA on the endoplasmatic
reticulum the tyrosinase expression is reduced.
Investigating the influence on tyrosinase
expression in melanoma cells
(B16V), we show that Belides (1%) is able
to reduce the biosynthesis of endogenous
tyrosinase to more than 50% (Fig. 3). Belides
thus avoids melanogenesis at a very
early stage.
Of course, these results also demonstrate
that expression of tyrosinase is not completely
blocked. Stronger inhibition or
even complete blocking of this expression
is not desirable for physiological
reasons. However, Belides exerts an additional
influence on the activity of remaining
amount of the enzyme. To assess
the inhibitory effect of Belides on
tyrosinase enzymatic activity, L-tyrosin
was incubated with mushroom tyrosinase
and the L-DOPAchrome formation
was measured. We could show that Belides,
in a concentration-dependent manner,
inhibits enzymatic activity which was
reduced by treatment with the active
(2.5%) to about 70% (Fig. 4).
Furthermore, the conversion of L-tyrosine
to melanin (Scheme 1) is an oxidative
process that requires the presence of
air oxygen. Other short-lived oxidizing
molecules - e.g. free radicals – namely
Fig. 3 Bioassay detecting the reduction of endogenous tyrosinase
expression in melanoma cells (B16V) after preincubation
with Belides (7 DIV). Tyrosinase activity was measured
in melanoma cells after removal of the active
reactive oxygen species (ROS) can also
promote melanin formation. An overproduction
of ROS in the cells or tissues
can alter biomolecules like L-tyrosine
and represents the so-called »oxidative
stress«. Antioxidants like polyphenols
can scavenge ROS and inhibit oxidation
of biomolecules like L-tyrosine. Belides
was shown to strongly inhibit formation
of one representative ROS, the superoxide
(O 2 .- ). To attribute the O2 .- scavenging
activity on an antioxidative principle,
Belides was first standardized to the
total polyphenol content. It could be
demonstrated that 34.2 µg/ml polyphenol
equivalents reduce the O 2 .- formation
to more than 50% (IC 50 = 29.13 µg/ml
polyphenol equivalents, Fig. 5).
To investigate if the skin-lightening effect
of Belides is only attributed to the
antioxidative capacity of the polyphenols
contained, it was determined the influence
on melanin synthesis in the presence
of a definite polyphenol amount of
arbutin (hydroquinone-mono-β-D-glucopyranoside)
in relation to equal polyphenol
amounts of Belides. Compared to
Belides arbutin is a single compound and
described in the literature as a tyrosinase
inhibitor and a widely used skin whitening
agent (13). When melanoma cells
(B16V) were exposed to 13,6 µg Belides
polyphenol equivalents/ml (7 DIV) the
melanin accumulation was reduced to
26.4% in comparison to the control cultures.
When arbutin was applied at the same
concentration (13.6 µg polyphenol equivalents/ml)
the melanin formation was reduced
only to 75.9%. The much stronger
effect of Belides point to other mechanisms
of action apart from tyrosinase inhibition
(Fig. 6).
Melanocytes as producers and distributors
of the pigment melanin play the key
role in pigmentation development. However,
these cells are not able to express
their own growth factors under nonstimulated
conditions. Therefore melanocytes
show a very low mitotic rate in
homeostasis (14).
Interaction with the surrounding cells,
like keratinocytes, Langerhans cells e.c.,
regulates complex biosynthesis processes
of melanogenesis by controlling
melanocyte growth and differentiation
via release of cytokines.
The peptide endothelin-1 (ET-1) is one
mitogen that increases melanin synthesis
as well as melanocyte proliferation.
The inflammatory mediator interleukin-1
(IL-1), which is induced by
UV irradiation in keratinocytes, increases
ET-1 expression in these cells tremendously.
It is also known that in Lentigo
senilis lesions a hypersecretion of ET-1
occurs (15-17). This fact directed us
to investigate the influence of Belides
Fig. 4 Bioassay measuring tyrosinase inhibition (mushroom
tyrosinase) using L-tyrosine as a substrate. Tyrosinase activity
was measured over L-DOPAchrome formation. Belides
concentration-dependently inhibits the tyrosinase enzymatic
activity in comparison to the control (maximum inhibition
= 100%)
44 SÖFW-Journal | 131 | 7-2005

COSMETICS
SKIN-LIGHTENING
on ET-1 expression. When keratinocytes
(HaCaT) where incubated with Belides
(1.0, 1.5 and 2%) and stimulated with interleukin-1
(IL-1) we could demonstrate
that Belides reduced the expression of
ET-1 to 31.8, 29.9 and 27.9% in comparison
to the control (100% expression).
The HaCaT cells used in these experiments
showed an expression of ET-1 under nonstimulated
conditions too, whereby Belides
was able to diminish the basal release
of ET-1 as well (Fig. 7).
Another factor that regulates melanogenesis
is the peptide α-MSH which interacts
via the melanocortin-1 receptor
(MC1-R). Nowadays it is known that this
peptide is not only produced in the pituitary
gland but also in the cells of the skin
(e.g. keratinocytes, melanocytes, Langerhans
cells). α-MSH can be upregulated
by UV irradiation and acts as a paracrine
as well as autocrine factor of skin pigmentation.
Therefore it is not a hormone
in the sense of the definition (18).
α-MSH binding to MC1-R enhances the
second messenger cAMP which in turn
activates tyrosinase and the production
of eumelanin (16, 19). To prove if Belides
influences the binding capacity of α-
MSH to the MC1-R we carried out an experiment
based on a competitive reaction
between a possible bonding of Belides
or the natural ligand α-MSH to
the MC1-R. The results obtained with
melanoma cells show that 1 and 1.5%
Fig. 6 Reduction of melanin formation. Belides is leading to
significantly less melanin production than arbutin.
Fig. 5 Analysis of the superoxide (O2 .- ) scavenging capacity of Belides. O2 .- was generated
by an enzymatic reaction of hypoxanthin and xanthinoxidase (XOD). The
assay is based on the spectrophotometric detection of formazan formation due to
the reaction of O2 .- with NBT in competition with the added Belides polyphenol
equivalents. Compared to the control (100% superoxide formation) Belides inhibits
O2 .- formation concentration-dependently (IC 50 = 29.13 µg polyphenol equivalents/ml,
standardized on gallic acid)
Belides decreases the binding capacity
of α-MSH to 38 and 12%. Therefore,
Belides has antagonist features inhibiting
the α-MSH binding capacity on the
MC-1 R (Fig.8).
On the other hand, cAMP also increases
rearrangement of actin filaments which
are among the major scaffolds of cell
structure and cell morphology. In consequence,
cAMP promotes melanocyte
dendricity - a ramification connecting
melanocytes with keratinocytes. This is
essential for the formation of the socalled
»epidermal melanin unit« which is
an association of one melanocyte and
surrounding 35 keratinocytes (18).
Fig. 7 Influence on endothelin (ET-1) expression. Human
epidermal keratinocytes (HaCaT), pretreated with Belides
(3 DIV), expressed lower amounts of ET-1 with and without
IL-1 stimulation (2 DIV) in comparison to control cultures
(with maximum ET-1 expression = 100%)
46 SÖFW-Journal | 131 | 7-2005
(µ

Fig. 8 Inhibition of α-MSH binding capacity on the MC1receptor.
Belides inhibited the alpha-MSH binding capacity
in melanoma cells (B16V) when the cultures were preincubated
with the active (3 DIV)
COSMETICS
SKIN-LIGHTENING
Fig. 9 Uptake of inert Fluospheres by keratinocytes (HaCaT)
after UV irradiation as a model of melanosome phagocytosis.
HaCaT cells incubated with Belides (3 DIV) irradiated with
UV light show a decreased uptake of Fluospheres in comparison
to control cultures (with 100% phagocytic activity)

COSMETICS
SKIN-LIGHTENING
To sum up, ET-1 as well as α-MSH control
dendricity and have an influence on
phagocytosis - the transfer of melanosomes
from melanocytes to keratinocytes.
In keratinocytes the melanosomes form
the secondary lysosomes around the keratinocyte
nucleus. Melanosomes in dark
skin differ from those in skin types I and
II, they are larger and packaged as single
units, whereas the melanosomes in fair
skin are smaller and packaged in groups.
During differentiation of keratinocytes
in the Stratum corneum the degradation
of the melanosome unit is much stronger
in skin types I and II than in dark skin,
which generates a kind of »melanin dust
effect« and make these skin types appear
less pigmented (20).
To study the influence of Belides on melanosome
transfer we used a Fluosphere
beads model. For simulation of melanosome
uptake HaCaT cells were irradiated
for phagocytosis stimulation with UV
light. It could be shown that 1.0 or 1.5%
Belides (1 DIV) significantly reduced the
Fluosphere uptake (25.9 and 40.9%
related to cell count), compared to control
cultures (set to 100% uptake), when
the phagocytic activity was monitored
(Fig. 9).
Furthermore, microscopic images taken
from control cultures and cultures preincubated
with Belides showed that a lower
number of FluoSphere beads were accumulated
around the cell nuclei when
the keratinocytes were treated with Belides
(Fig. 10).
The in vitro results demonstrate that Belides
acts as a functional ingredient at
different stages of melanogenesis. By influencing
pathways of action before, during
and after melanin synthesis Belides
effectively reduces skin pigmentation.
Finally, to demonstrate that Belides produces
a noticeable lightening effect on
human skin an initial in vivo study was
carried out on 5 volunteers from the
Philippines (age: 19-39; 4 females, 1 male)
over a period of 4 weeks.
The results show a significant lightening
effect in comparison to the untreated
and placebo areas when oil-in-water formulations
(O/W) containing 2 or 5% Belides
were applied twice daily (Fig. 11).
Already after 14 days of application a
tremendous effect could be observed.
Skin lightening was increased to 20.3
and 30.8% while after 28 days to 19.3
and 31.5% (p < 0.05 versus untreated).
These results show that the whitening
process is complete already after 14 days
and continued application of the active
does not further lighten the skin. Moreover,
a complete knock-out of melanogenesis
could be risky for melanocyte
survival. Therefore, Belides acts as a gentle
modulator of skin pigmentation.
Fig. 10 Microscopic images showing (a) the Fluosphere uptake (red FluoSphere beads,
1.0 µm size) in control and (b) in Belides (1%) treated HaCaT cultures (1 DIV).
For visualization of the phagocytosis cells were counterstained with DAPI (blue
nuclei). Picture images were taken using a Leica DMIL fluorescence microscope
(Leica, Germany; PHAGO 3 oil lense 100 x, excitation filter: 515 - 560 nm)
Fig. 11 Efficacy test on human skin (pilot study) demonstrate the skin-lightening
activity of Belides (chromameter readings). Formulations were applied by 5 volunteers
twice daily on the inner forearm. Values are related to initial conditions
(p

COSMETICS
SKIN-LIGHTENING
� Conclusion
Belides has proven itself as a very efficient
active that successfully interferes
with every stage of the melanin synthesis.
The extraordinary composition of this
active helps to remove blemishes in a
very gentle and reliable manner. Belides,
a water-soluble active produced
without any organic solvent, meets the
ever-growing demands of natural beauty
care.
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