Soliance - StimulHyal

Skin elasticity enhancer

INCI China approved – Ecocert & Cosmo

StimulHyak – Product File – June 2015 1

Summary
Introduction ....................................................................................................................................... 3

StimulHyal.......................................................................................................................................... 4

Mechanism of action .......................................................................................................................... 5

Tests .................................................................................................................................................... 6
Increase in CD44 by immuno-labelling ......................................................................................... 6
Stimulation of hyaluronic acid and elastin production ................................................................. 7
Stimulation of the expression of the HAS2 gene ...........................................................................8
Improvement of biomechanical properties of the skin .................................................................11

Biodegradability and ecotoxicology ................................................................................................. 12

Characteristics .................................................................................................................................. 12

Cosmetic uses ................................................................................................................................... 12

Conclusion ........................................................................................................................................ 13

StimulHyak – Product File – June 2015 2

Introduction
High molecular weight Hylauronic Acid (HA) plays plays a key
role in essential biological pathways of the skin, and more
specifically in the extracellular matrix of the dermis. It helps to
support the matrix by forming a viscous network of polymers
through collagen fibers, and maintains hydration thanks to its
hygroscopic properties.

Hyaluronic acid is important in skin Hyaluronic acid

physiology for cell renewal and cell
migration. It maintain the biomechanical
properties of the skin.
Soliance has worked in biotechnology
processes for more than 20 years.
Thanks to its unique technical expertise,
Soliance has developed a new anti-aging
active ingredient specifically targeted to
improve skin suppleness and elasticity.
StimulHyal is a pure molecule. Its structure is
directly responsible for its activity. StimulHyal
acts in the dermis where many biological
activities take place.
It improves the skin biomechanical properties,
especially skin elasticity.
Molecules like HA and elastin are essential to
ensure cutaneous suppleness and are the main
targets of StimulHyal.
Its efficacy is proven by in vitro, ex vivo, in vivo
tests.
Ecodesign and sustainability arre the driving
force of Soliance product development.
Our production process has been optimized to
obtain StimulHyal, a product approved by
Ecocert.
Cutaneous aging: dermis matters
The extracellular matrix of the dermis (ECM)
The ECM of the dermis is made of a complex of
molecules, which give the skin its biomechanical
properties.
Among these components, collagen is
responsible for skin firmness, while hyaluronic
acid and elastin are key components of skin
elasticity.
The Hyaluronic acid molecule was isolated in
1934 by Meyer and Palmer. It is an essential
component of numerous tissues and physiological
fluids.
It is a linear biopolymer, the main component of
the glycosa- minoglycan family (GAGs).HA is a
natural biopolymer formed of repeated
disaccharidic units. These are D-glucuronic acidic
and D-acetylglucosamine, linked together by
glycosidic bonds β (1-3) and β (1-4).
The formula is (C14H20NNaO11)n with
200<n<700.
The skin contains more than 50% of the total HA
amount present in the body. It is mainly
synthesized by fibroblasts in the dermis, but also
by keratinocytes in the epidermis.
Hyaluronic acid is synthesized via the Hyaluronan
synthases HAS1, HAS2 and HAS3, a family of
enzymes related to the inner surface of the
plasma membrane.
These proteins catalyze the elongation of a chain
of HA by the addition of an alternating glucuronic
acid and N-acetylglucosamine.
Although these three isozymes are able to
synthesize hyaluronic acid, it was shown that
HAS2 is the major synthase and can catalyze the
synthesis of hyaluronic acid of high molecular
weight (2.106 Da).
Enzymes catalyze the length of the HA chains by
adding alternatively a glucuronic acidic and a N-
acetylglucosamine.
The extension of the de novo synthesized
polymer occurs in the cytoplasm. Almost 75% of
the HA is immediately exsuded in the cell.

StimulHyak – Product File – June 2015 3

Elastin
Elastin is another essential component of the
dermal ECM. It is a 64 to 66 kDa protein, mainly
constituted of glycine, valine, alanine and
proline. It gives the skin its elasticity and
suppleness, thus enabling it to recover its normal
state after deformation.
Epidermis
Dermis
During the ageing process, elastin and HA
synthesis tends to decrease, while certain
enzymes increase their degradation.
Consequently, the skin loses elasticity and
suppleness.
Soliance has developped a new active ingredient
that aims to fight skin ageing signs.

Skin histology

Hyaluronic acid molecule

StimulHyal
Product origin and characteristics Vegetable and renewable biomass are used both
StimulHyal is the trade name of calcium 2- as substrates and for bioconversion.
ketogluconate. This molecule is a highly purified This process is approved by Ecocert (soon
white powder (>95%). This calcium salt is Cosmos). It enables the formulation of ecological
readilysoluble in water until 10 g/L. cosmetic products.
StimulHyal is a natural molecule of a lactic  Natural molecule
 Biomass valorisation
 Solvent-free fermentation
HO OH
 Low energy cost process
O
2+  Ecocert and Cosmos
HO Ca
-
HO O
O
2

Calcium 2-cetogluconate StimulHyal Powder

bacteria metabolism.
 White powder
 Odourless
 Preservative free
 Very high purity Fermentation devices

Manufacturing process
Soliance has developed its expertise in white
biotechnology for 20 years and thus could adapt
the metabolism of lactic bacteria to produce
StimulHyal. The process is eco-friendly. No
solvent is used during fermentation, and the
product is obtained by physical treatments
(ultra-filtration, concentration and crystallisation)
in aqueous phase and at room temperature.

StimulHyal’s manufacturing process

StimulHyak – Product File – June 2015 4

Mechanism of action

StimulHyal helps to prevent the skin from

sagging and formation of smile lines

Improvement of the skin

biomechanical properties:
 Increase of the skin elasticity
 Decrease of the skin fatigue

 Stimulation of elastin synthesis in the dermis

 Stimulation of the synthesis of high MW HA in 1
the dermis via the hyaluronan synthase
overexpression 2 (HAS2)

StimulHyak – Product File – June 2015 5

Tests
Interaction with CD44
In silico test
CD44 is a glycoprotein cell receptor With advancing age, the body produces less and
involved in various different biological less hyaluronic acid, which has major
processes. It particularly plays a role in cell repercussions on CD44 receptor activation.
adhesion and migration. Maintenance of the three-dimensional structure
of the tissues is subsequently reduced.

Soliance has studied the interaction that

StimulHyal may have with this receptor. By
means of an in silico approach conducted in
partnership with the University of Reims
Champagne-Ardenne (URCA), the CNRS and the
Multi-scale Molecular Monitoring Platform (P3M),
it is shown that StimulHyal has the same affinity
Modelling of CD44 for the CD44 receptor as hyaluronic acid.

CD44 is the specific receptor for hyaluronic acid; Interaction between CD44 and reconstructed
it binds by the N-terminal globular end of the StimulHyal.
receptor, activating the latter.

Interaction of CD44 with hyaluronic acid

crystallized on the right and reconstructed on
the left. StimulHyal

This affinity therefore allows stimulation of the

CD44 receptor.

StimulHyal activates the CD44 receptor in

CD44 Hyaluronic acid silico and displays a "HA-like" effect.

Increase in CD44 by immuno-labelling

Ex vivo test
An ex vivo test is performed in order the The nuclei are stained with propidium iodide.
supplement the results of the in silico study. Image analysis allows quantification of the area
occupied by the CD44 receptors.
Protocol
Phase Ingredient %
Some explants derived from an abdominoplasty
of a Caucasian woman of 39 years of age are Hydroxyethyl cellulose 1%
A
used for this study. Aqua QSP 100%
A gel containing 0.5% StimulHyal is applied to
B Preservative QS
the explants at a rate of 2mg/cm2 on D0, D2 and
D4. The controls do not receive any C StimulHyal 0.5%
treatment.The explants are frozen on D0 and D5.
The CD44 receptors are immunolabelled with an D Alcohol 96 25%
anti-CD44 monoclonal antibody. Formule used

StimulHyak – Product File – June 2015 6

Results
On D5, the pharmaceutical form containing **
StimulHyal induces a significant increase of
239% in the area occupied by the epidermal **

% area occupated
CD44. 2,4
StimulHyal stimulates and increases CD44
receptors.
Percentage area occupated by CD44 in the
epidermis
Control
0.7

D0 0.2

DEJ
D0 D5
Student’s statistical test Control
** p<0,01 Very signficiant
Control StimulHyal 0.5 Gel containing 0.5% of Stimulhyal
%

D5

CD44
immunolabelling

Stimulation of hyaluronic acid and elastin

production
Ex vivo test
Protocol
Fibroblasts from abdominal biopsy of a 33 years-
old woman are incubated:
 24 h at 37 °C, under moist atmosphere and 5
% of CO2 for hyaluronic acid assessment
 96 h at 37 °C, under moist atmosphere and 5
% of CO2 for elastin stimulation assessment.
Incubation is processed :
 Alone (control)
 or with a positive reference,
 or increasing concentrations of StimulHyal.
Positive references used in the study are :
 for hyaluronic acidic production : Transforming
Growth Factor β(TGF-β), at 10 ng/ml,
 for elastin production : ascorbic acid, at 100
μg/ml .
The statistic significancy between « Control »
conditions and « Reference product », was
assessed by a Student t-test. The statistic
significancy between «Witness » conditions and «
Tested product », was assessed for each product,
by a variance analysis using One Way ANOVA,
followed, when necessary, by a Holm-Sidak test
(* : p<0,05).
Results
StimulHyal is a pure molecule, its activity is
directly related to its structure.
Remark : the difference between the results
obtained with StimulHyal at 10µg/ml and at
1000µg/ml are not significant.
The results obtained for the stimulation of elastin
and hyaluronic acid synthesis by StimulHyal are
patented.

At the end of the incubation, proteins contained StimulHyal significantly stimulates elastin and
in cell lysates are quantified by hyaluronic acid synthesis.
spectrocolorimetric method (Bradford method).
Results are compared to the total amount of
proteins.

StimulHyak – Product File – June 2015 7

Hyaluronic acid synthesis
StimulHyal significantly stimulates hyaluronic
acid production even at 100 µg/ml with a dose-
dependent effect
Stimulation of the expression of the HAS2 gene
In vitro test
Contrary to the other methods, qRT-PCR
(quantitative reverse transcription
polymerization chain reactions) allows
quantification of trace amount of RNA in a
sample. RNA results from the transcription
of a gene, a DNA fragment, and is the
template for protein synthesis.
Principle of qRT-PCR
This methods first consists in a reverse
transcription (RT) of the RNA into the
complementary DNA. Secondly, the cDNA is
duplicated on big scale by PCR for several cycles.
The polymerized DNA quantity is measured in
real time.
Interest of qRT-PCR
By quantifying the RNA present in a sample, it
can reveal the increased expression of a target
gene, equally a raise of the production of HAS2,
that is the major enzyme involved in the high
molecular weight HA synthesis, therefore, a
specific response from the cell.
Protocol
1. Fibroblasts derived from skin explants
obtained from a 25 years old woman are
incubated 24 hours at 37°C in a humid
atmosphere and 5% CO2.
StimulHyak – Product File – June 2015
Elastin synthesis
StimulHyal significantly stimulates elastin
production even at 10 µg/ml with a dose-
dependent effect.
The explants are incubated either in a culture
medium alone or in the presence of StimulHyal
at 100 µg/ml.
2. The total RNA was extracted from cells grown
in 6-well plates, using 0.5 ml of Extract-all
(Eurobio) per well. After extraction, the quantity
was measured by absorbance at 260 nm and
RNA quality was assessed by migration on
agarose gel.
3. Two hundred fifty nanogrammes of total RNA
are used for the synthesis of complementary
DNA (Verso SYBR Green 2-Step Kit,
ThermoElectron).
4. The complementary DNAs synthesized are
subjected to the PCR cycles according the
following process :
 95°C during 10 seconds (cDNA denaturing)
 60°C during 1 minute (selected primers
hybridization on cDNA brains then primers
elongation into amplicons*)
5. The amplicons number is calculated in real
time (at each cycle) measuring the absorbance
at 260nm.
8
 Results
qRT-PCR results are normalized with respect to 2
reference genes (RS18 and RPL32) using the
"DDCt", whose the expression is unchanged.

The tests were conducted by Fanja Rabenoelina

and Laurent Duca UMR CNRS / URCA 6237
"Extracellular Matrix and Cellular Dynamics
(MEDyC) Laboratory SIRM (Signals and receptors
matrix).

* DNA neosynthesized from primers

Statistical result of qRT-PCR products Qualitative result on agarose gel

The expression in the control sample is set at The measurement of RNA present in the cells
1 for relative expression. treated with StimulHyal allows us to show the
enhanced expression of the gene HAS2.
The statistical significance of differences between
the terms “Control" and "StimulHyal, was
assessed by a t test (Student t-test. *, P <0.01).
StimulHyal at 100 µg /ml enhances the
expression of the gene HAS2 by 60% versus
Migration of
control, verified by the quantification of the
HAS2 at its
genes duplicates. molecular
weight

Témoin
StimulHyal 100
µg/ml

This enhanced expression is validated by the

appearance of a spot which is more intense than
the control (i.e. not treated with StimulHyal)
after migration of the qRT-PCR products
(including HAS2) on agarose gel according to
their molecular weights.

StimulHyak – Product File – June 2015 9

StimulHyal at 100 µg/ml improves significantly StimulHyal significantly stimulates the production
the production of the enzyme HAS2, major actor of high molecular weight hyalu-ronic acid via the
in the production of high molecular weight HA. enhanced expression of the gene HAS2.

Stimulation of HA production in the dermis

Ex vivo test
Protocol
Six abdominal biopsies from a Caucasian 39 Hyaluronic acid is the main acid GAG found in the
years-old woman are prepared and incubated in skin. The increase of the blue color induced by
an specific medium BEM (BIO-EC’s Explants StimulHyal 0.5% is directly related to the
Medium). hyaluronic acid synthesis in the dermis.

At D0, D2, D4, a cream containing 0,5% of
StimulHyal is applied on 3 biopsies, at 2 mg/cm2.
The 3 control are not treated.
At D5, the 3 biopsies of each samples are treated
the same way.
The cream containing 0,5% of StimulHyal
induces a significant increase of 183 % of the
surface occurred by Alcian blue coloration in the
papillary dermis, compared to the control
samples at D5.

After 48 h of fixation in ordinary Bouin solution, We may conclude that StimulHyal stimulates the
samples are dehydrated and impregnated into production of hyaluronic acid in the dermis.
paraffin with a Leica 1020. They are fixed into
blocks with a Leica 1160, and 5 mm-slices are
cut with a microtome Minot, Leica RM 2125 and
then fixed on glass histological slides
Superfrost®.

Acid GAGs are observed after coloration with

Alcyan blue into the whole papillary dermis. In
this zone, the GAGs (acid ones) appear in blue.
Image analysis enables us to quantify the
coloration intensity, which is directly linked to
hyaluronic acid synthesis stimulation. Results are
statistically treated with a Student-t test.

StimulHyal significantly stimulates hyaluronic

acid production in the dermis
Control skin explant Skin explant treated with
(untreated skin) at D5 0,5% StimulHyal at D5

StimulHyak – Product File – June 2015 10

Improvement of biomechanical properties of the
skin
In vivo test
Protocol
A panel of 22 Caucasian female volunteers (age Placebo StimulHyal
mean is 43 years old) apply twice a day, on half Ingredients
cream cream
of the face for 28 days :
 A cream formulated with 0.5% of StimulHyal Xyliance 4% 4
 A placebo cream Caprylic/capric
12% 12
triglyceride
The skin elasticity was evaluated at D14 and D28
using a cutometer (MPA580 Courage & Khazaka). Aqua QSP 100% QSP 100%

Preservative qs qs
Skin elasticity
At 28 days, the cream formulated with 0.5% of StimulHyal 0 0,5%
StimulHyal significantly increases skin elasticity:
+ 16% versus placebo.

Results
In vitro results are confirmed. StimulHyal
improves skin elasticity within 28 days

Skin fatigue
The cutaneous fatigue corresponds to the skin’s
ability to recover its initial shape after
undergoing successive distortions (the skin is
sucked up 3 times by the negative pressure of
the cutometer probe). By decreasing skin
fatigue, we help it to restore its initial state.

At 28 days, the cream formulated with 0.5% of

StimulHyal significantly decreases skin fatigue: -
27% versus placebo.

StimulHyal significantly decreases the skin

fatigue within 28 days

StimulHyak – Product File – June 2015 11

Biodegradability and ecotoxicology
Biodegradability Ecotoxicity
StimulHyal is labelled as easily bio-degradable, StimulHyal is not ecotoxic, regarding the OECD
with respect to final bio-degradability assessment test on Daphnia sp.
of the OCDE 301F directive. CL50 (48h) > 10 000mg/L

StimulHyal is an eco-responsible ingredient

Characteristics Cosmetic uses

StimulHyal INCI Name is Calcium ketogluconate With age, the skin loses its elasticity, face
expressions becomes more visible. By limiting
Aspect: Powder cutaneous fatigue, StimulHyal helps the skin to
maintain its tone and smoothes wrinkles out.
Color: White to off-white
Skin sagging causes the face contour to become
Preservative: None less defined. StimulHyal restores skin elasticity
and suppleness in order to redefine the oval of
Recommended dosage: at 0.3% - 0.5% in a the face, neck and décolleté.
wide range of cosmetic products.
Lips too lose volume, elasticity and firmness
Skincare Benefice Products while ageing. By stimulating HA and elastin
synthesis, StimulHyal re-shapes the lips, plumps
Day cream
and smoothes them for a younger-looking smile.
Anti ageing : Night cream
Elasticity
Age defying Decolleté and
Suppleness After pregnancy, or weight loss, the skin gets
Mature skin neck specific
zone area care loose. StimulHyal helps to re-shape the body.

Day cream
Elasticity
Anti strechmark
Suppleness
Bodycare care
Comfort
Body resculpting
Skin aspect
cream

Day care
Comfort and
flexibility Anti-stretch
appearance of
Elasticity mark treatment
the skin
resculptants care

Active make Elasticity

Foundation
up Suppleness
StimulHyak – Product File – June 2015 12
Conclusion
Soliance is a cosmetic ingredient supplier with years of expertise in white
bio-technology. StimulHyal is a new active molecule obtained through
this technology.

StimulHyal is a pure molecule whose performance is proven by

biological tests (in vitro, ex vivo, in vivo). The results confirm the
stimulation of hyaluronic acid synthesis in the dermis. StimulHyal also
stimulates elastin production and limits skin fatigue.
Results are visible within 28 days.

StimulHyal is:
Preservative-free
White powder with no odour
Ecocert and cosmos approved
Eco-designed and produced through a eco-friendly manufacturing
process
Ecoresponsible
Patented
REACH-compliant

StimulHyal is an innovative active cosmetic ingredient with proven

performance. It helps the skin to restore its biomechanical properties
such as elasticity and suppleness.

StimulHyak – Product File – June 2015 13