Active Ingredients
RenovHyal, a Patented Anti-Ageing Cosmetic Ingredient
Authors: Celina Rocquet, Romain Reynaud, Soliance, France
Abstract
Because of its barrier function, the skin is of prime importance
for separating the interior of the body from the outside. Therefore,
it reflects what happens inside: our health, our mental well-being
and our age. Chronological ageing is responsible for progressive
skin structure degradation. As a result, moisturisation and
cutaneous firmness decrease, thus weakening this organ.
Wrinkles form and tonicity decreases dramatically.
In order to fight this irreversible phenomenon, we have
developed and patented an original cosmetic active ingredient:
RenovHyal, that allies hyaluronic acid’s fantastic properties to
an innovative biological action.
Skin ageing concerns both the dermis and the epidermis. In the
epidermis, the barrier function is altered. The consequence is
an increase of Trans Epidermal Water Loss (TEWL). During this
phenomenon, water evaporates at the surface. The skin is thus
less moisturised, loses its flexibility, tonicity and softness.
The epidermis is formed by cohesive keratinocytes, living a life
cycle of 28 days, while they differentiate from the basal
towards the surface. Cell cohesion is essential to regulate this
mechanism and to ensure cell renewal and differentiation. Cells
are thus related one to another by cell junctions. Different types
of junctions exist with multiple but specific roles: sealing, signal
communication, chemical transmission. Among them, tight
junctions are essential for skin barrier integrity, ensuring cell
cohesion. They enable keratinocytes to form a natural
functional barrier in the stratum granulosum, leading to water
flow regulation and TEWL limitation.
The dermis is mainly constituted of fibroblasts and the
extracellular matrix (ECM), where collagen, elastin and
glycoaminoglycans such as hyaluronic acid may be found.
Hyaluronic acid is of paramount importance in skin
moisturising and collagen, synthesised by fibroblasts is
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necessary for skin support. Skin ageing causes an increase in
their degradation and a decrease in their synthesis. The
consequence is a loss of suppleness and flexibility, leading to
wrinkle formation and dehydration.
RenovHyal is an Ecocert-approved low molecular weight
hyaluronic acid (15.000 and 50.000 Da) strictly identical to
the molecule present in the skin. RenovHyal is produced from
plants through fermentation using renewable plant resources
that mimic natural processes. Our company has almost 20
years of know-how in the production of hyaluronic acid.
RenovHyal enables the skin to maintain skin homeostasis. On
the one hand, it reinforces tight junctions between
keratinocytes, thus limiting transepidermal water loss.
Moreover, thanks to its natural hygroscopic properties, it forms
a “water reservoir” in the dermis. On the other hand, firmness
is remarkably improved by RenovHyal. Efficacy tests
demonstrate that this specific grade of HA improves
procollagen I synthesis in the dermis. The skin is firmer, better
moisturised, younger.
Introduction
The skin is the first cell layer in contact with the
environment.
Because of its barrier function, the skin is of prime importance
for protecting the organism from external aggressions (sun,
extreme temperatures, infections). Chronological ageing is
responsible for progressive skin structure alteration.
Consequently, moisturisation and cutaneous firmness decrease,
thus weakening this organ. Wrinkles are formed and tonicity
decreases dramatically. In order to fight this irreversible
phenomenon, we have developed and patented an original
cosmetic ingredient: RenovHyal. It allies hyaluronic acid’s
fantastic properties with innovative biological action.
RenovHyal enables the skin to maintain an optimal hydration
level, as well as homeostasis. It is therefore an exceptional
cosmetic active ingredient to fight skin ageing signs.
Cosmetic Science Technology 2008
 Active Ingredients
Skin Ageing
Skin ageing results from multiple biological processes occurring
both in the dermis and the epidermis. In the epidermis, the
barrier function of the skin is altered, leading to an increase of
trans epidermal water loss (TEWL). This natural phenomenon
results from the diffusion of water from blood circulation
towards dermis and the different cell layers of the epidermis.
The water finally evaporates at the surface. The skin is
therefore less moisturised, loses its flexibility, its softness and
firmness. In the dermis, the amount of molecules responsible
for supporting the skin structure (collagen, elastin, hyaluronic
acid) decreases. Their synthesis process decreases while their
degradation process increases.
Epidermis, hydration and cell junctions
The epidermis is formed by multiple layers of cohesive cells
called keratinocytes. These cells have a 28-day life cycle while
they differentiate and are “pushed” towards the most external
part of the skin by the basal membrane. Cohesion is essential
in order to assure cell renewal and differentation.
These cells are linked one to another by junctions. Different
types exist, with different roles, such as waterproofness, as well
as channels for chemical signal transmission. Three main
groups of cell junction have been described to link epithelial cell
and participate to the cell communication (Lodish et al., 2005):
• Anchoring junctions bound cells one to another and enable
the skin to assure rigidity and solidity. Desmosomes, adhesive
junctions and hemi-desmosomes belong to this class.
• Gap junctions (or nexus) are responsible for intercellular
communication. They are constituted of bound connexines,
forming transmembrane channels that link the cytoplasm of
two adjacent cells. Small molecules and ions may go
through gap junctions, leading to metabolic and energy
coupling of adjacent cells.
• Tight junctions (or zonulae occludentes) are in charge of
cell waterproofness. They are mainly localized in the
stratum granulosum.
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The main biological functions of tight junctions are:
- Binding neighbouring cells, thus ensuring cell cohesion.
They form a natural functional barrier (Tebbe et al., 2002)
- Regulating extracellular water flow and limiting trans
epidermal water loss (TEWL)
- Jamming proteins and transmembrane lipid diffusion in
the plasmic membrane, which contributes to the epithelial
cell polarity.
Two constitutive proteins of these junctions have been
described: ZO-1 and occludin. They are expressed in mature
keratinocytes, in injured skin, and in vitro in differentiating
keratinocytes (Pummi et al., 2001). A scientific study has
evaluated the expression of these proteins during re-
epithelisation and cell regeneration of the stratum corneum.
Their role is also necessary in epidermis renewal as it has
been described by Malminen, (2003).
Tight junctions play a fundamental role in maintaining the
integrity of the skin barrier, in epidermis hydration and in
its organization.
Adhesion belt
Tight junctions
Desmosome
Epidermis
GAP junction
Keratinocytes
Hemidesmosome
Dermis DEJ
Basal lamina
Figure 1 Multiproteic cell junctions in the epidermis
Intercellular junctions ensure mechanical and chemical
cohesion between cells, but also cell communication. Ensuring
these physiological activities helps to prevent skin ageing.
Cosmetic Science Technology 2008
 Active Ingredients

Dermis, hyaluronic acid and collagen
Our skin is naturally subject to chronological ageing. One of
the visible manifestations is the loss of firmness and flexibility,
mainly due to the decrease of structural molecule quantity,
such as collagen, elastin or glycosaminoglycans (GAGs).
ECM and hyaluronic acid
The extracellular matrix (ECM) is formed by all the
components present in the extracellular space of the dermis.
It is mainly synthesised by fibroblasts and plays the role of
interstitial medium. The extracellular matrix enables the
dermis molecules and cells to make contact with the basal
membrane, which is a junction surface between the dermis
and the epidermis. It is a reservoir of essential molecules for
cell survival and adaptability, such as growth factors. Different
types of molecules are present in the ECM:
- Proteins: Collagen, elastin
- Glycoproteins : fibronectins, laminin
- Glycosaminoglycans and proteoglycans: hyaluronic acid,
the extracellular medium. Some adhesion receptors present at
the surface of the cells bind the HA for a better solidity of the
dermis. Hyaluronic acid is one of the main constituents of the
ECM. Due to its exceptional physical properties, it plays a
predominant role in the structure and organisation of the
dermis, and helps to ensure flexibility and firmness of the skin.
Because of its high water retention ability, hyaluronic acid is of
prime importance in skin moisturising regulation. It has been
proved that hyaluronic acid amount decreases with age.
Mature skins are altered at different levels, such as loss of skin
elasticity, wrinkles and dehydration, intimately linked to HA
quantity reduction. HA is the essential molecule in the
biological process of hydration regulation.
Collagen and fibroblasts
Collagen is present in many organs, in different types.
Scientific literature currently describes 28 types of collagen.

sulfate chondroitin, aggregan, perlecan. GAGs are linear Type I collagen – the most abundant in the Human body, may

polymers composed by repetitive disaccharidic units.
Hyaluronic acid (HA) is the most abundant GAG. Synthesised
by fibroblasts thanks to a specific enzyme bound to the
plasmic membrane (HA synthetase), it is directly secreted in
be found mainly in skin, healing tissues, tendons, and in some
parts of bone tissue. Collagen is a protein present in the
extracellular matrix and conjunctive tissues that are
synthesised by fibroblasts. It represents 25% of total proteins
in animals, and the main protein of the ECM.

Type Form Molecular Structural Tissues

composition characteristics

I Fiber [α1(I)]2[α2(1)] Fibrils (300 nm) Skin, tendons, ligaments,

dentine, interstitial tissues

III Fiber [α1(II)]3 Fibrils (300 nm) Skin, muscles, blood vesssels

Fibrils (390 nm) with a N-

V Fiber [α1(V)]2[α2(V)], terminal globular Skin, smooth muscle, teeth,
[α1(V)]3 extensions often with the bones, cornea, placenta
type I

IV Lamellar [α1(IV)]2[α2(IV)] Bidimensional network All basal layer

VII Lamellar [α1(VII)]3 Long fibrils Under basal layer in the skin

XIII Transmembrane [α1(XIII)]3 Intra-Membrane protein Hemi-desmosomes in the skin

XVII Transmembrane [α1(XVII)]3 Intra-Membrane protein Hemi-desmosomes in the skin

Table 1 Different collagen types present in the skin (Lodish et al)

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 Active Ingredients

Collagen structure is made of three α polypeptidic chains parallel, an increase of degradation processes of collagen

of repeated units of Glycine, Proline and Hydroxyproline.
Procollagen α chains are synthesised (as every protein) in
the endoplasmic reticulum (1). Oligosaccharides are
added to the C-terminal propeptide (2). Thus formed
propeptides join to form trimers that are linked covalently
by disulfide bounds (3).
Procollagens are fold down and transported in the Golgi
apparatus, where lateral association of the chains lead to
fascicles (4). They are then secreted (5) and the
propeptides are cut (6). Trimers join into collagen fibrils,
that are then covalently bound (7). These fibrils may then
form bigger structures called collagen fibres.
by specific Matrix Metallo Proteases (MMPs) occurs.
Consequently, with time, elasticity and firmness of the
skin decrease with age. The face contour blurs, eyelids
sag, the skin weakens, gets thinner and less hydrated.
Expression lines appear, wrinkles too. It is thus very
important to improve collagen I synthesis, which
represents almost 80% of dermis collagen.
RenovHyal: A Patented Active Ingredient
RenovHyal is a low molecular weight hyaluronic acid in a
sodic form whose weight is between 15.000 and 50.000
Da. The primary structure of hyaluronic acid is constituted
by a repetitive disaccharide unit of sodium glucuronate and

Collagen is the support protein of the dermis. In aged N-acetyl glucosamine, linked by a (1-3) bond.

skin, a decrease of collagen synthesis is observed,

compared to younger skin (Varani et al, 2000). In See Figure 3: Chemical structure of hyaluronic acid overleaf

Fibroblast Synthesis Degradation

Endoplasmic
reticulum
C
1 C

N C ECM
N
N
N N N
2
Golgi appartus
HPS47
4
3
Procollagen
(triple helix)

5
Cytoplasm

Collagen fibril

MMPs Peptides
7 Collagen fibre (collagenases) Amino acids

Figure 2 Synthesis and degradation processes of collagen

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 Active Ingredients

COONa
CH2OH
O
H O
H O H
O H
OH H
H H
OH H
H OH
H HNCOCH3
n

CH 3
O
C
HO
NH
OH O
O O HOCH2
HO O
C O O
HO O
O O HOH2C

OH NH C
O O
HO O C

CH3

Figure 3 Chemical structure of hyaluronic acid

RenovHyal Production directly in the epidermis and even in the dermis in order to

Our company produces hyaluronic acid through a biomimetic regulate hydration and to increase skin firmness. RenovHyal

process. For years, we have been continuously improving an
original process of the sodium hyaluronate through
fermentation of lactic bacteria and a plant renewable
substrate. RenovHyal is derived from natural and renewable
materials only. Plant-derived substrates are used in the
process, such as wheat or beetroot, in order to supply the
necessary elements for bacteria growth (glucose, nitrogen and
mineral salts). Bioethanol is used for precipitation. Thanks to
an environment-friendly process that respects Ecocert
specifications, RenovHyal is an Ecocert approved ingredient
for the cosmetic industry.
normalises hydration due to an innovative and patented mode
of action. It limits trans epidermal water loss by reinforcing
tight junctions between keratinocytes of the stratum
granulosum. We have proved that RenovHyal stimulates the
expression of ZO1 and Occludin proteins, that are
constitutive proteins of tight junctions. This improves cell
cohesion and the barrier function of the skin. Moreover,
thanks to the high hygroscopic ability of the hyaluronic acid,
it maintains hydration in the skin through a “water reservoir”
function in the dermis. Through both of these
complementary actions, RenovHyal regulates the internal
hydration of the skin.

Mechanism of action of RenovHyal We have also demonstrated that the specific grade of
RenovHyal is a 15.000 - 50.000 Da hyaluronic acid. This RenovHyal remarkably increases cutaneous firmness by
specific molecular weight enables it to enter the skin and act stimulating collagen I in the dermis.

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 Active Ingredients

TEWL

r
ction einforc
jun e Skin hydration

m
ht

ent
Tig
Epidermis

Stimulation of tight junction protein synthesis

Tight junction

Trans Epidermal Water Loss (TEWL) prevention

DEJ

retentio
ater n"
H 2O "W

HA hygroscopic characteristics
H2O
HA Water retention in the dermis
Dermis

nthesis Skin mechanical properties

1 sy in
en
cr
gla

ea
Col

Fibroblast Stimulation of collagen I synthesis

se

Increase of skin firmness and hydration

Collagen

Figure 4 RenovHyal mechanism of action

Stimulation of tight junction constitutive protein
synthesis
It has been proved that ZO-1 and Occludin proteins are
constitutives of tight junctions between keratinocytes. The
test aims to prove that Renovhyal stimulates both of these
protein synthesis, by measuring ZO-1 and Occludin quantity
after application of Renovhyal. This experiment has been
processed on normal human keratinocytes (NHK) stemming
from plastic surgery.
Protocol
Human keratinocytes were seeded into 12 well-plates in a SFM
medium (Invitrogen 17005075) and incubated for 24 hours.
After incubation, the culture medium was removed and
replaced by culture medium containing or not containing
(control) the compound at selected concentrations. Cells were
incubated for 72 hours (37ºC,CO2., 5%). The experiment was
performed in triplicate. Membrane proteins were extracted into
RIPA buffer. The proteins were placed in wells (3,6 µg/well)
and separated through a SDS-PAGE gel electrophoresis. After
migration, proteins were transferred on a nitrocellulose
membrane. Non specific sites of the membrane were
saturated (overnight at 4°C) in a fat free milk saturation buffer
PBS/0.05%/5% (PBSTM). After washing in PBST (PBSTM
without milk), specific antigenic sites were labelled with a
monoclonal antibody: anti-Occludin or anti-ZO1. After
incubation (1 hour at room temperature) and washing in PBST,
each antibody was revealed with a peroxydase-conjugated
secondary antibody. The peroxydase activity was revealed using
Enhanced Chemiluminescence method (ECL). The analysis of
the relative intensity of the ZO-1 and Occludin bands is
expressed in OD and measured by the One-D-scan software.

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 Active Ingredients

Results
+39% +29%
8 0.8

7 0.7
Protein quantity (DO)

Protein quantity (DO)

6 0.6

5 0.5

4 0.4

3 0.3

2 0.2

1 0.1

0 0.0
Control Renovhyal Control Renovhyal
5mg/ml 5mg/ml
Occludin ZO-1

Conclusion (Procollagen Type I C-peptide (PIP) EIA kit - MK101, Takara).

Renovhyal stimulates ZO-1 (A) and Occludin (B) synthesis. Each cell well was dosed in duplicate. Therefore, 8 OD
values were obtained for each condition. The obtained
Collagen I is the most abundant protein of the dermis. It is optical densities (OD) for the standards were reported on a
synthesised by fibroblasts and is involved in skin firmness. logarithmic graph. Known PIP (ng/ml) concentrations were

The test aims to prove that RenovHyal increases collagen I reported as abscissa and OD as Y-axis. Measured OD for

synthesis by measuring type I pro-collagen quantity (PIP) after each sample (supernatant) were reported on the graph. PIP

application of RenovHyal. The test has been processed on concentration for each tested sample, was determined in

aged human fibroblasts stemming from plastic surgery.
Protocol
Normal human fibroblast culture was performed for 24 hours
and subject to tested product treatment for 48 hours.
Supernatants containing procollagen I were collected and
stored at –20°C until the dosage step. The tested product was
RenovHyal at 5 mg/ml. Vitamin C was also tested as a positive
control of procollagen I synthesis enhancer, because of its
cofactor role in the reaction. Cells were treated such that each
concentration of the product was evaluated in 4 wells of NHF.
Dosage of procollagen I was processed through the measure
of the protein quantity contained in cell supernatant
ng/ml. A protein dosage was performed on the cell pellet.
For each cell well and each treatment condition, a
quantitative dosage of PIP release in ng/ml was obtained.
This dosage calculated versus a cell quantity was
expressed in µg. The final result was then expressed in
ng/ml of PIP / µg de proteins. Dosage of the proteins
present in the cell pellets after treatment of the cells with
the product was processed. It is expressed in µg of cell
proteins and related to PIP dosage for each cell well. The
method principle is similar to the Lowry method. The
stimulation percentage was calculated as follows, with the
quantitative values of PIP related to proteins: ([produced
PIP] – [control PIP] x 100. A Student test is calculated.

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 Active Ingredients

Results Conclusions
Dehydration and loss of firmness are signs of skin ageing that
*p<0.01 +21% lead to wrinkle formation and sagging of some areas of the
110 face. The use of RenovHyal helps to maintain a moisturised
and firm skin by improving cell cohesion and collagen
synthesis. The skin is therefore progressively redensified and
100 *
moisturised. It restores skin tonicity. The skin is younger, nicer,
both flexible and firm. RenovHyal is an exceptional cosmetic
90
ingredient because it is:

• effective
P1P/Prot

80
• 100% Natural
• biomimetic
70
• obtained from plant-derived renewable resources
• patented
60 • approved by Ecocert.

50
Control Renovhyal
5mg/ml
Conclusion
Renovhyal significantly increases procollagen I synthesis.
Evaluate the effect on biomechanicals properties of a cream
containing Renovhyal 0,5% vs placebo. The test was
performed on 22 voluntary women, between 50 and 60
years old. The average age of the panel was 54. The
voluntary women have loose skin on the face.The volunteers
applied twice a day for 56 days, a cream containing 0.5%
Renovhyal. At the end of the study, they auto-evaluate the
product. After 56 days of use, results showed that the panel
considered that:
- The skin quality is improved : 91% of the panel,
- The skin smoother : 87% of the panel,
- The skin is better moisturized : 91% of the panel,
- The skin is firmner 91% of the panel,
- The skin tonicity is improved : 87% of the panel
Cosmetic Applications
RenovHyal may be incorporated in different kinds of cosmetic
lines of products such as preventive age-defying skincare,
firming products, after sun products, after shave products and
liquid make up.
References
1. Pummi K, Malminen M, Aho H, Karvonen SJ, Peltonen J,
Peltonen S (2001) Epidermal tight junctions: ZO-1 and Occludin
are expressed in mature, developping, and affected skin and in
vitro differenciating keratinocytes. Journal of Investigative
Dermatology. Vol 117 (5): 1050.
2. Malminen M, Koivukangs V, Peltonen J, Karvonen SL, Oikarinen
A, Peltonen S (2003) Immunohistological distribution of the tight
junction components ZO-1 and occluding in regenerating human
epidermis. British Journal of Dermatology. Vol 149 (2): 255.
3. Tebbe B, Mankertz J, Schwartz C, Amasheh S, Fromm M, Assaf
C, Schultz-Ehrenburg U, Sanchez Ruderish H, Schulke JD,
Orfanos CE (2002) Tight junction proteins : a novel class of
integral membrane proteins. Expression in human epidermis and
in HaCaT keratinocytes. Arch. Dermatol. 294 (1-2): 14-18.
4. Varani et al (2000) Vitamin A antagonizes decreased cell groxth
and elevated collagen-degrading matrix metalloproteinsas and
stimulates collagen accumulation in naturally aged human skin.
J. Invest. Dermatol. 114: 480-6.
5. Lodish, Berk, Matsudaira, Kaiser, Krieger, Scott, Zipursky, Darnell
(2005) Biologie Moléculaire de la cellule, 209-30.
Authors’ Biographies
Celina Rocquet has a Master of Science in Molecular Biology, Plant
Physiology and Biotechnology. She specialized in Marketing through a
Master of Business degree. After working in the cosmetic industry
(SIPCA/Adedis, Clariant, LVMH-Parfums Christian Dior) and in a
marketing and communication agency (Publicis), she became
Marketing Manager at Soliance in 2006.
Romain Reynaud has a Master of Science in Formulation Chemistry. He
has been working in ARD (Agro Industrie Recherche et Développement)
for three years, before becoming R&D Manager at Soliance.

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