TECHNICAL

FILE

ACTIVE
INGREDIENTS

HYDROSELLA™
Osmo-hydration to fight against dry skin
Organic extract of Wild Rosella selected for its high content in Betaine

Stimulates organic osmolytes cellular intake and restores skin barrier function by
increasing lipid and tight junction proteins production

Provides rapid and continuous improvement of skin hydration and reduces skin
water loss up to 72 hours after application

Proven efficacy in rinse-off applications

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TABLE OF CONTENTS
Table of Contents ............................................................................................................................... i
SUMMARY ......................................................................................................................................... 1
INTRODUCTION ................................................................................................................................. 2
Hydrosella™: Osmo-hydration to fight against dry skin .............................................................. 10
EFFICACY STUDIES - In vitro ............................................................................................................. 15
EFFECT OF HYDROSELLA™ ON ORGANIC OSMOLYTES AND THEIR TRANSPORTERS ................... 16
EFFECT OF HYDROSELLA™ ON CELL VOLUME REGULATION ....................................................... 18
EFFICACY STUDIES – Ex vivo ............................................................................................................ 22
EX VIVO EVALUATION OF HYDROSELLA™ ON ORGANIC OSMOLYTE TRANSPORTERS ................ 23
EX VIVO PROTECTION FROM DELIPIDATION BY HYDROSELLA™ ................................................. 26
CLINICAL STUDIES ............................................................................................................................ 36
CLINICAL EVALUATION OF HYDROSELLA™ ON DRY SKIN: FAST AND LONG-LASTING ACTION ... 37
CLINICAL EVALUATION OF HYDROSELLA™ ON THE AGRESSED SKIN OF ACTIVE PEOPLE ........... 40
CLINICAL EVALUATION OF HYDROSELLA™ MOISTURIZING EFFECT IN RINSE-OFF APPLICATION45
CONCLUSION ................................................................................................................................... 49
COSMETIC APPLICATIONS ............................................................................................................... 50
RECOMMENDED USE....................................................................................................................... 51
ADDITIONAL DATA ........................................................................................................................... 52
EFFECT OF HYDROSELLA™ ON INFLAMMATION IN NORMAL HUMAN DERMIS FIBROBLASTS ... 53
REFERENCES..................................................................................................................................... 55

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SUMMARY
INCI NAME Glycerin (1) (and) Water (2) (and) Erythritol (3) (and) Hibiscus Sabdariffa
Fruit Extract (4)
CAS 56-81-5 (1), 7732-18-5 (2), 149-32-6 (3), 84775-96-2 (4)
EINECS 200-289-5 (1), 231-791-2 (2), 205-737-3 (3), 283-920-7 (4)
ORIGIN Organic botanical extract of Australian Wild Rosella.
Hydrosella™ is a natural extract from a unique variety of Hibiscus
(Hibiscus sabdariffa). Organically grown in Australia, this unique
genotype has been selected for its high betaine content.
COSMETIC PROPERTIES • Based on the natural osmosis system
• Protects and fights against drying-induced cell shrinkage
• Increases organic osmolyte receptors
• Increases organic osmolyte intracellular content
• Accelerates keratinocyte water intake
• Induces the production of lipid synthesis
• Improves tight junctions in skin
SKIN BENEFITS / • Rehydrates dry skin cells
CLAIMS • Provides an immediate and long-lasting moisturizing effect
• Improves skin barrier function to further reduce water loss
APPLICATIONS • Daily skin care • Regenerating care
• Face care • Anti-aging care
• Body care • Skin repair treatment
• Smoothing care • Spa care
• Dry skin care • Men's skin care
• Moisturizing care • Protective care
• Hydration care
RECOMMENDED 1-2%
DOSAGE
PH RANGE USE 4.0 to 8.0
INCORPORATION Should be incorporated at the end of the formulation at a temperature
below 40°C. However, Hydrosella™ is stable when heated up to 6 hours
at 80°C. To prevent or limit color changes during heat stability, the
addition of a chelating agent and an antioxidant is recommended.
INCOMPATIBILITIES To date, no incompatibility has been observed.

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INTRODUCTION
The skin is known to be the largest organ of the human body. One of its primary functions is to
protect against external environment aggressors such as bacteria, microorganisms, chemicals, etc.
This barrier also helps to regulate and prevent the loss of fluids and electrolytes that are important
in regulating the human body1. Therefore, keeping skin healthy is of upmost importance so it can
fulfill its protective role.

One of the important conditions for skin to be healthy is hydration. When skin gets dry, it becomes
less resilient when faced with environmental irritants. Increased skin dehydration can cause skin
to peel or crack, leading to even greater skin dehydration and water loss, allowing contaminants
to easily penetrate the body. In addition to the reduced physiological role of protection,
dehydrated skin looks dull, rough and even flaky2, tends to lose its smoothness, suppleness and
can worsen the appearance of wrinkles, aging and conditions such as inflammation.

Dry skin (xerosis) is a well-known condition and can affect people of all ages. According to US
Pharmacist, it is estimated that 75% of people above the age of 64 suffer from dry skin3. In most
cases, dry skin is not a medical concern and does not represent a serious problem. However, it
leads to skin discomfort, constant itchiness, skin roughness or a feeling of skin tightness.

Moreover, it can be difficult to treat, as it is influenced by many different factors:

• Low humidity;
• Conditioned air;
• Cold temperatures;
• Surfactants such as soap;
• Exposure to chlorine (pool);
• UV exposure;
• Cleansing routines;
• Medication, diet, lifestyles, clothing
• Chronological aging.

Knowing that dry skin can affect anyone all year long and is caused by various factors, it's no
surprise that moisturizing, or hydrating, is one of the most sought-after benefits and product
staples for most consumers, including men.

According to Mintel’s Trends and Opportunities in Moisturizing Facial Care Insight4:

• 72% of US women use a facial moisturiser – as do 27% of men
• 75% of female skincare buyers in China use a facial moisturiser or similar in the morning
and 64% do so in the evening

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• 39% of UK men use a moisturiser, making it the second most popular product after
cleansers
• From March 2018 to February 2019, 61% of face/neck care products claimed to be
moisturizing or hydrating. This is the second biggest claim, surpassed only by the
botanical/herbal origin claim (66%).

Well-hydrated skin results in greater benefits than just skin comfort. In 2018, In-Cosmetics Asia
announced that hydration was one of the three key things to focus on to bring skin closer to a
glass-like, clear appearance, and was an important pathway to skin that is healthier, plumper,
more supple and bright. “Ingredients that provide hydrating properties are pivotal to the
development of new products that help deliver pure, clean, radiant and glowing skin” 5 . Many
cosmetic companies agree, suggesting that skin should be moisturized every day to maintain a
fresher and more radiant look.

Dry skin process

Skin is composed of approximately 64% of water6, and the epidermis itself is approximately 70%
water7. Because water is such an important component of skin for its function and properties, the
human body has developed its own mechanisms to maintain suitable hydration levels and protect
against external elements. The epidermis, the outermost of the skin's layers, is regarded as the
most important skin layer in maintaining hydration. However, with today’s way of life, constant
exposure to heated or conditioned air, pollution etc. the
skin's natural mechanisms for staying hydrated are
overwhelmed.

The epidermis is composed of 5 layers:

1. Stratum corneum: outermost layer of skin,
mainly made of cornified cells, keratin and lipids;
2. Stratum lucidum: thin layer with a lighter
appearance;
3. Stratum granulosum: made up of highly
differentiated keratinocytes migrating from the
stratum spinosum becoming known as granular
cells;
4. Stratum spinosum: composed of keratinocytes;
5. Stratum basale: deepest epidermal layer,
attaches the epidermis to the basal lamina,
below which lie the layers of the dermis.

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The dry skin process starts at the stratum corneum (SC), the
outermost layer seen as the main barrier to water loss. The SC
is described as a “Bricks and Mortar” 8 model to show the
importance of its structure made of corneocytes, embedded
cells in a lipid matrix (ceramides, cholesterol, fatty acids). It
can absorb as much as five to six times its own weight9.

Regular exposure of the skin to soap, rough clothing, hot

shower/bathing routine, chlorine from pools and hot or cold
weather can cause lipid depletion. The “disappearance” of the
lipid barrier breaks down the integrity of the cutaneous barrier (bricks and mortar), increasing skin
flaking and peeling and increasing skin dehydration as it leaves the barrier open.

Evolution of delipidation process of the Stratum corneum and breakage of the cornified cells layer.

Just beneath the SC is the stratum granulosum (SG) in which we find keratinocytes at their final
differentiation stage before becoming corneocytes. Keratinocytes act as a second barrier to help
maintain skin hydration and to protect against microbial, viral, fungal and parasitic invasion. They
also play a key role in protecting against UV radiation and minimizing heat. Following the breakage
of the first skin barrier (SC), extracellular water will exit the epidermis into the external
environment. This will reduce the extracellular water content in the deeper layer of the epidermis
and create a hypertonic environment for the keratinocytes. In response to the hypertonic
environment, intracellular water content will be removed from keratinocytes through osmosis,
leading to cell shrinkage. Constant cell volume is important in maintaining cellular activity. To do
so, cells such as keratinocytes will use various strategies to regulate their volume and help retain
or regain cell volume,10. Among the various strategies, organic osmolytes are seen as an optimal
strategy as this also influences the tight junctions’ barrier and lipids secretion of the SC 10,11,12.
Organic osmolytes are small, naturally-occurring organic, water-soluble compounds.

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Moisturizing strategies
Cosmetic ingredients for hydration/moisturizing can be divided into three categories13.

1- OCCLUSIVES

Occlusive ingredients hold water in the epidermis, they act as a

protective seal over the skin, preventing it from losing water.
They are usually oily, fatty or waxy substance such as lanolin,
lecithin, mineral oil, paraffin, and stearic acid.

2- HUMECTANTS

Humectants act like sponges that pull water in from deep skin
layers and from the air to help keep the surface skin layer
(stratum corneum) hydrated and moist. Among the humectants
are ingredients such as glycerin, honey, panthenol, propylene
glycol, aloe vera gel, lactic acid, polymeric polyols such as
polydextrose and sugar alcohols. However, humectants should
be used carefully as they could potentially make skin even drier
by pulling water into a damaged stratum corneum that can no
longer hold hydration.

3- EMOLLIENTS

Emollients simply fill in the cracks between skin cells (brick and mortar model) without adding
moisture. They help the skin feel smooth, but they don’t impact skin hydration. Some emollients
can play a double role and also act as humectants. Emollients are compounds such as vegetable
oils, beeswax, almond oil, petrolatum, zinc oxide, paraffin,
mineral oil and glycerin.

Most actual strategies do not work at the cellular level with

mechanism such as osmosis.

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Osmosis
Osmosis is defined as a natural process by which the
molecules of a solvent tend to pass through a semi-
permeable membrane from a less concentrated solution into
a more concentrated one, thus equalizing the concentrations
on each side of the membrane. In biology, osmosis is the
movement of water or osmolytes out of or into the cell to
balance the concentrations on each side of the cellular
membrane. Osmosis happens spontaneously.

Osmolytes or solutes are compounds affecting osmosis. They are soluble in the solution within a
cell, or in the surrounding fluid. They can be inorganic (e.g. Na+, K+, Cl-) or organic.

TONICITY

Tonicity is a measure of the osmotic pressure gradient between two solutions separated by the
cell membrane. In other words, tonicity is the relative concentration between the solution on each
side of the cell membrane. It will affect the movement of water from on side of the cell to the
other.

Isotonic (Iso = same, Tonic = concentration) means that the

concentration of solutes (molecules) is the same on each
side of the cell membrane.

Hypertonic (Hyper = more, Tonic = concentration) means that the

concentration of solutes (molecules) is higher outside of the cell.

Hypotonic (Hypo = less, Tonic = concentration) means that the

concentration of solutes (molecules) outside of the cell is lower than
inside the cell.

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In biology, if a cell is placed in a hypertonic (high concentration) solution, water will leave the cell,
and the cell will shrink. In an isotonic (equal concentration) environment, the relative
concentrations of solute and water are equal on
both sides of the membrane. There is no net
water movement, so there is no change in the
size of the cell. In a hypotonic (low concentration)
environment, the concentration of solutes is
greater inside the cell than outside of it, the cell
will absorb water to dilute its internal
concentration14.

Organic osmolytes
Organic osmolytes have been widely studied in nature as they are used by cells of numerous
organisms and tissues to maintain cell volume in response to a stress. In insects, organic osmolytes
stabilize membranes at sub-zero temperatures15 . Marine animals and organisms use them to
match the osmolarity of their surroundings (e.g. highly salted water) and to counteract the effect
of hydrostatic pressure. Even in mammals very rarely exposed to high osmolarities, we do find
organic osmolytes. The highest concentration of organic osmolytes is in renal medullary cells in
response to high NaCl and urea concentration. In some plants, organic osmolytes such as betaine
are produced during periods of drought to retain water, avoid dehydration and protect their cells
from breakage16.
Production of Organic
Hyperosmotic stress or
osmolytes and
UV
transport

Keratinocytes lose
Keratinocytes intracellular water Improved cell volume
and shrink recovery

Organic osmolytes can be divided into three groups17:

1- Polyols (e.g. sorbitol and myoinositol);
2- Amino acids and their derivatives (e.g. taurine, alanine, proline);
3- Methylamines (e.g. betaine, glycerophosphorylcholine).

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In nature, the main purpose of organic osmolytes is to help cells regain their volume and water
content. However, they have been shown to have other advantages:
1- Also called compatible solutes, they have been shown to regulate cell volume without
causing any damage to cell membranes or cellular function.
2- They help stabilize proteins and protect them from degradation, unlike inorganic ions such
as Na+, K+, and Cl-18.
3- Recent studies have shown that organic osmolytes can improve the skin's barrier function
by stimulating the secretion of lipids and tight junction proteins such as occludin10, 12.

BETAINE

Betaine is a very well-characterised organic osmolyte from the methylamine family. It is found in
many living species and has been widely studied in plants. Plants producing more betaine have a
higher tolerance to dryness or environmental stresses during their life cycle19. One of the reasons
for this increased tolerance is because betaine effectively stabilizes the structure of various
enzymes and proteins and helps to maintain the integrity of cell membranes against the damaging
effects of a high salt environment, cold, heat and even freezing20. Also, when applied topically on
leaves, betaine is rapidly absorbed by leaf tissues and helps restore cell water content 21 . In
mammals, betaine is commonly found in renal cells where it counteracts the effects of urea and
helps stabilize enzymes and proteins22.

Organic osmolytes and betaine are also found in human keratinocytes. Evidence shows the
existence of a concentration gradient of organic osmolytes and their transporter starting at the
stratum spinosum, with the highest concentration found in the stratum granulosum. Betaine and
other organic osmolytes are synthetized in keratinocytes following an osmotic stimulus or UV
exposure to protect keratinocytes from cell damage, maintain protein integrity and restore cell
volume23,24.

TIGHT JUNCTIONS AND ORGANIC OSMOLYTES

The skin barrier plays in important role in the regulation of water loss. Lipids filling spaces between
corneocytes have been widely noted and are now commonly referred as the “brick and mortar”
model. As explained earlier, the loss of lipids can accelerate water loss. Like lipids, “tight junctions”
(TJ) have an equally important role in “sealing” the gaps between cells and preventing further
water loss. Tight junctions control inter-cellular permeability and the diffusion of water and
solutes. They are transmembrane proteins (e.g. occludin, claudin-1) found in the uppermost part
of the stratum granulosum7.

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Following UV or osmotic stress, keratinocyte volume decreases, and the secretion of TJ is greatly
reduced, damaging the skin barrier and reducing skin permeability further. To maintain a proper
skin barrier, keratinocyte volume, TJ and lipid content should be restored. Recent studies
demonstrate that organic osmolytes such as betaine can accelerate keratinocyte volume recovery,
increase lipid production and restore TJs such as occludin, demonstrating the important role
played by organic osmolytes in skin hydration and restoring the skin barrier10.

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Hydrosella™: Osmo-hydration to fight against dry

skin
What if we could restore skin hydration in a new innovative, never-before-seen way? What if
instead of using an occlusive barrier, we could help cells regain their water volume more rapidly
and more efficiently, increase lipid secretion and tight junctions for a better skin barrier that
provides a complete hydration response? Lucas Meyer Cosmetics has found the solution: a new,
natural Australian active ingredient with multi- target action for improved skin hydration.

Origin of Hydrosella™
Hydrosella™ is an organic extract of the calyx of a unique genotype of
Hibiscus sabdariffa L. specially selected for its high naturally- occurring
betaine content. Known in Australia as Wild Rosella, it is thought to have
originated in Sri Lanka and was introduced to Australia by fishermen and
traders thousands of years ago. It has now naturalized in the tropical
regions of Australia where drought periods are common.

This wild hibiscus is an annual crop. It can grow up to 2

meters high and 1 meter wide. It is considered as a hardy spice for its
ability to tolerate drought. The hibiscus flower is a symbol of delicate
beauty. The calyx surrounds the flower and protects it from drought and
environmental stress during its development. Without the calyx, the
flower wouldn’t be able to reveal its beauty.

COMPOSITION OF HYDROSELLA™

Hydrosella™ is Cosmos Organic-certified. It uses non-palm certified organic glycerin, certified

organic Wild Rosella and organic non-GMO erythritol from a corn starch source.

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Traditional usage
Hibiscus sabdariffa has been traditionally cultivated for food and medicinal
properties. The calyxes are served fresh or dried in herbal drinks, hot and
cold beverages, fermented drinks, wine, jelly etc. The leaves can be steamed
for making chutney or eaten as salad. They are dried during harvesting
season to be used during the off-season.
Various parts of the plants have been used traditionally for medicinal or
therapeutic purposes to treat colds, toothaches and other infections25,26.

Common uses
Nowadays, hibiscus is still very popular in the food industry. The petals can be used
for making jellies and dessert garnishes, adding their unique flavour. The calyxes
are used in jelly or syrup that can be added to champagne or white wine to add a
sophisticated touch to the drinks.
Because of its unique phytochemical content of polyphenols, anthocyanins,
polysaccharides and organic acids, hibiscus is said to have many health benefits,
including calming fevers, soothing an upset stomach and fighting bacterial
infections.

Sustainability of Hydrosella™
Our unique variety of Wild Rosella is sourced from a small, family-owned
business situated near Coffs Harbour on the North Coast of New South
Wales. The hibiscus plantation is approximately one hectare in size with
around 2,000 plants developed from seed sourced from wild naturalized
populations in the Kimberly region of North West Australia.

Plants are drip irrigated with water from a nearby spring-fed dam.
The Wild Rosella is certified organic, so weeds are controlled using mulch and mechanical
methods, i.e. slashing. The harvest period is typically in March, with annual planting conducted
from November to January at the latest. The plantation is bounded to the west by Moonee Creek
and to the north, west and south by a large section of NSW State Forest. Being a fairly passive

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operation, the growing and harvesting of Wild Rosella has negligible
impact on the waterway and surrounding vegetation. The
harvesting period utilizes a small local workforce.

Our grower has many years’ experience working as a sustainable

agriculture facilitator for the Australian Government, and due to this
experience, he strives to achieve an environmentally and socially
conscious business. The undertaking is considered sustainable as
there is minimal resource use or impacts to the natural
environment.

Our Phyto-Cross™ extraction process is

water-based and does not use synthetic chemicals or produce
hazardous waste. The efficient technologies used in the extraction
phase consume little energy and maintain the integrity of the botanical
active components. To ensure high-quality extraction and batch-to-
batch consistency, Hydrosella™ is standardized in betaine content
coming from wild rosella. No chemical betaine is added.

Our production facility purchases 100% renewable energy, thereby

contributing to our goal of reducing greenhouse gas emissions. All liquid
and solid waste generated from the production process is collected, treated, and turned into a soil
conditioning product (circular waste).

The certified organic glycerin used in Hydrosella™ is sourced from a company

that provides development opportunities for farmers by way of training in
sustainable and organic farming techniques such as crop selection for
climatic conditions, maintenance of soil fertility, crop rotation to minimize
pests and diseases, and the use of microorganisms to reduce environmental
impact. The glycerin is naturally derived from soy, maize and other annual
crops. There are no solvents used in the manufacturing process.

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Erythritol
Erythritol is a natural humectant for personal and skin care applications. It is naturally-occurring
in foods such as grapes, melons and honey, as well as in fermented foods such as cheese and wine.

As a humectant, erythritol was added to Hydrosella™ to increase the available water content in
the inter-cellular space of the epidermis to allow faster water intake by the keratinocytes following
the stimulation of organic osmolytes and their transporters.

The erythritol used is derived from corn starch and sourced from a company with ISO
certifications:
• ISO 9001: Quality management
• ISO 14001: Environnemental management

It has SA8000, which is a certification standard that encourages organizations to develop, maintain
and apply socially acceptable practices in the workplace, meaning no child or forced labour, safe
working places, freedom of association and non-discrimination.

The erythritol is certified under Ecocert and Ceres standards, which are compliant with EU organic
regulations.

Osmo-hydration to fight against dry skin

Hydrosella™ is standardized in betaine, a natural organic osmolyte from the Wild Rosella.

Hydrosella™ offers a unique mode of action, fighting the root cause of skin dehydration by
stimulating the body's natural response to dryness using a dual approach to provide a complete
solution. It stimulates natural osmolyte strategy of keratinocytes and simultaneously restores skin
barrier function by:
1. Stimulates TauT (taurine transporter) on keratinocytes;
2. Increases organic osmolytes intracellular content;
3. Reduces keratinocytes water loss while increasing and accelerating volume recovery;
4. Improves inter-corneocytes neutral lipids synthesis in curative and preventive manner to
help restore skin barrier organization;
5. Stimulates tight junction protein occludin which act as a solute barrier to reduce water
loss;
6. Increases Stratum corneum thickness in a laminated aspect representative of well-
hydrated skin.

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EFFICACY STUDIES - IN VITRO

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EFFECT OF HYDROSELLA™ ON ORGANIC

OSMOLYTES AND THEIR TRANSPORTERS
INTRODUCTION
Given the skin's constant exposure to environmental stressors (e.g. UVR, conditioned air, etc.), the
epidermis is under pressure to maintain its water content. To do so, keratinocytes adopt a variety
of strategies. It has been shown that organic osmolytes have an important role to play24.

Organic osmolytes, also known as compatible solutes such as taurine and betaine, are of great
interest in skin hydration and protection because of their involvement in cell volume homeostasis
and protection, i.e.: against oxidative stress. For cell volume recovery, the osmolyte strategy
requires the expression of specific osmolyte transporting systems such as the TauT transporter27.

OBJECTIVE
The aim of the study was to evaluate the effect of Hydrosella™ on the synthesis of osmolyte
transporters and osmolyte intracellular content in a model composed of normal human
keratinocytes with quantified taurine and TauT content.

PROTOCOL
TESTED PRODUCTS
Hydrosella™ was tested at the following concentrations: 0.05%, 0.1% and 0.2%.
Hydrosella™ betaine content at 0.2% is 4 µM.
Betaine was tested at 4.3 mM as a positive control, in a separate study.
PMA (phorbol myristyl acetate) at 10ng/mL was used as reference activator of taurine transporter
synthesis.

To evaluate the activity of both components of Hydrosella™, Wild Rosella extract and erythritol
were tested separately. Wild Rosella was tested at 0.2% (erythritol part replaced by water),
erythritol at 0.02% and Hydrosella™ at 0.2%.

BIOLOGICAL MATERIALS
Normal human keratinocytes were obtained from a 50 years old donor for Hydrosella™
evaluation, 76 years old donor for betaine evaluation and 62 years old donor for the comparison.
Keratinocytes were cultivated in monolayer culture until reaching confluency.

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METHOD
Cells were incubated for 24 hours in the absence (control) or in the presence of increasing
concentrations of Hydrosella™.

EVALUATION
Both taurine transporters (TauT) and taurine intracellular content were quantified in cell lysate
using a specific and sensitive ELISA kit. Total proteins contained in the cell lysates were quantified
using a spectro-colorimetric method (BRADFORD).
Results are expressed as ng of TauT per mg of total proteins and as ng of taurine per mg of total
proteins (mean ± S.D.).

STATISTICAL ANALYSIS
Level of significance between “Control” and “Test Compound” has been assessed independently
for each product by a one-factor variance analysis (One-way ANOVA) followed by a Holm-Sidak
test (*: p<0.05, **: p<0.01, ***: p<0.001).

RESULTS

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Hydrosella™ increases organic osmolytes transporters TauT and organic osmolytes intra-cellular
content. It demonstrated a higher efficacy than pure betaine showing the synergistic activity of
the natural extract.

To confirm that the activity on the intra-cellular content of organic osmolytes was coming from
the Wild Rosella, a comparative study was made. Erythritol alone do not significantly stimulates
organic osmolyte production while a Wild Rosella extract was able to increase it by 44%.

CONCLUSION
Hydrosella™ significantly increases both organic osmolyte content and osmolyte
transporters in keratinocytes

Wild Rosella extract is responsible for the increase of intracellular organic osmolytes
content

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EFFECT OF HYDROSELLA™ ON CELL VOLUME

REGULATION
INTRODUCTION
Cell volume regulation is necessary for water homeostasis and the preservation of cell function.
These regulatory mechanisms are vital in many tissue types across living organisms since excessive
cell shrinkage or swelling can lead to cell death 28 . The movement of water across the cell
membrane is impacted by solute concentration in the cell and of the surrounding environment,
which in turn determines the cell volume. In the skin, tightly controlled cell volume regulation is
one aspect that contributes to the skin's hydration status24. These mechanisms respond to any
changes in osmotic pressure from physiological stressors that impact the cells. One such cell
volume regulation mechanism involves naturally-occurring compounds called organic osmolytes.

The cell volume regulation tests were made by The University of Manchester

OBJECTIVE
The aim of this project was to investigate the ability of Hydrosella™ to modulate function using
live cell imaging.

PROTOCOL
TESTED PRODUCTS
Hydrosella™ was tested at a concentration of 0.2%.
PMA (phorbol myristyl acetate) at 10ng/mL was used as reference activator of TauT transporters
synthesis.

BIOLOGICAL MATERIALS
Primary normal human epidermal keratinocytes (NHEKs) from 3 different donors were cultured
according to the manufacturer’s instructions (PromoCell).

METHOD
Single cell live-imaging
NHEKs were seeded at single cell density and treated with Calcein green for visualisation. Live cell
imaging was carried out over a 35 minutes cycle during which NHEKs were exposed to 5 minutes
in iso-osmotic conditions, 15 minutes in hyperosmotic conditions and 15 minutes in iso-osmotic

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conditions. This was carried out in the absence and presence of Hydrosella™ at 0.2%
concentration. Relative cell volume, percentage cell volume regained and rate of regulatory
volume increase (RVI) mechanism were then calculated from these videos.

STATISTICAL ANALYSIS
Level of significance between “Control” and “test compound” has been assessed independently
for each product by a two-way test t of Student for paired data. The statistical significant value is
p<0.05. (*: p<0.05, **: p<0.01 and ***: p<0.001).

RESULTS
Single-cell live imaging was used to elucidate whether Hydrosella™ has an effect on cell volume
regulatory mechanisms in primary keratinocytes. Firstly, when exposed to hyperosmotic
conditions keratinocytes undergo cell shrinkage and regain some cell volume when returned to
iso-osmotic conditions. This is improved in the presence of Hydrosella™. The percentage of cell
volume regained was analyzed when the cells were returned to iso-osmotic conditions following
the hyperosmotic exposure.

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The rate of regulatory volume increase (RVI) was analyzed from when the cells were returned to
iso-osmotic conditions following hyperosmotic stress. The rate of RVI is define as the speed at
which the cell regains its volume. Hydrosella™ enhances the velocity of cell volume recovery in

primary keratinocytes compared to control.

Hydrosella™ at 0.2% improved the percentage of cell volume regained following hyperosmotic
stress.

CONCLUSION
Hydrosella™ improves human keratinocytes cell volume regulatory mechanisms, thus
improving water homeostasis control

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EFFICACY STUDIES – EX VIVO

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EX VIVO EVALUATION OF HYDROSELLA™ ON

ORGANIC OSMOLYTE TRANSPORTERS

OBJECTIVE
The aim of this study was to confirm ex vivo the effect of Hydrosella™ on osmolyte transporter
expression.

PROTOCOL
TESTED PRODUCTS
Hydrosella™ was tested at the following concentration: 0.4%. Betaine content in the Hydrosella™
samples is evaluated around 5 µM at a dosage of 0.4%.
Betaine was also evaluated at the concentration of 5µM and 20µM.

BIOLOGICAL MATERIALS
Explants were prepared from an abdominoplasty on a 43 and 44-year-old Caucasian female.

METHOD
The explants were placed alive in BEM culture medium at 37°C in a humid, 5%-CO2 atmosphere.
After 24h, the culture medium was replaced with serum-free DMEM containing 1% antibiotics and
1% amino acids for approximately 8 hours. Betaine and Hydrosella™ were then added for an
additional 48 hours.

EVALUATION
TauT immunostaining was carried out on skin sections using a monoclonal anti-TauT antibody
diluted at 1/100 in PBS – 1% BSA and revealed with an Alexa 488 secondary antibody (green
labelling). The immunostaining was performed manually and assessed by microscopical
observation.

IMAGE ANALYSIS
Image processing was performed using Fiji software 29 . We used a DAPI dye (blue channel) to
identify nuclei in the epidermis region. The green channel was used to quantify fluorescence
labelling as a result of the TauT expression in the epidermis region. Mean TauT intensity was then
computed as a ratio between epidermis total intensity and area size.

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The results are reported as percentage of total fluorescence intensity compared between the
control and the treated assay. Obtained data and percentage variations were submitted to two-
way t test of Student’s for paired data. The statistical significance value is p<0.05 (*p<0.05,
**p<0.01, ***p<0.001).

RESULT
All pictures are at zoom 40x. Blue label corresponds to the nuclei, and green label to TauT
expression.

Epidermis

Untreated Betaine 20μM Hydrosella™ 0.4%

Nuclei and TauT Nuclei and TauT Nuclei and TauT

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The results demonstrate that Hydrosella™ increases the production of the osmolyte transporter
TauT. With Hydrosella™, the increase is higher than compared to equivalent pure betaine,
showing a synergetic action from the extract.

CONCLUSION

Hydrosella™ significantly increases osmolyte transporters, helping the natural osmolyte

defense of the skin

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EX VIVO PROTECTION FROM DELIPIDATION BY

HYDROSELLA™
INTRODUCTION
The barrier function of the stratum corneum (SC) is largely dependent on specific lipids filling
intercellular space between corneocytes. Decrease of skin lipid content occurs early with aging
and is aggravated by unfavourable environmental conditions, leading to enhanced transdermal
water loss, in addition to dryness and scaling of the skin.

Delipidation (removal of lipids or lipid groups) of skin explants offers a convenient model for
hydration study. Lipid removal leads to a compaction of the SC, representing a dehydration state.
This modification can be clearly observed using lipid immunostaining. Therefore, this model allows
us to test hydration and skin barrier improvement in the presence of Hydrosella™ for either its
preventive or curative action.

OCCLUDIN IMMUNOSTAINING:

Tight junctions are a multiprotein complex composed of transmembrane proteins including

occludin, claudins and adhesion molecules, associated with other proteins such as ZO-1, ZO-2 and
ZO-3. Occludin is a 65 kDa completely membrane protein and the first component of tight
junctions described. Occludin can interact with zonula occludens-1 (ZO-1) for the skin barrier
function. In the epidermis, occludin is localized essentially in the stratum granulosum. Occludin is
a good marker of barrier function integrity.

NEUTRAL LIPIDS STAINING

LipidTOX™ neutral lipid stain is a fluorescent dye that has an extremely high affinity for neutral
lipid droplets and can be detected by fluorescence microscopy or an HCS reader.
In the skin, many neutral lipids can be stained, such as phospholipids, cholesterol, cholesteryl
esters, ceramides, triglycerides, etc.
The stratum corneum is the most extensively studied component of the barrier. This highly
impermeable structure is the result of keratinocyte differentiation and consists of highly
keratinized dead cells embedded within a lipid matrix. The appropriate deposition of lipids in the
extracellular spaces and their correct organization into bilayer membranes (forming intercellular
lipid lamellar structures) is an important factor for the formation of an effective barrier to TEWL.
Many studies have shown that the correct ratio of ceramides, cholesterol and free fatty acids is
crucial for the formation of lamellar structures30.

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OBJECTIVE
The aim of the study is to evaluate both the preventive and curative action of Hydrosella™ against
cellular dehydration on human living skin explants ex vivo.
This activity has been assessed by:
• Cellular viability analysis
• Immunostaining of occludin
• Immunostaining of neutral lipids by LipidTox® probe.
• Immunostaining of organic osmolyte transporter (TauT)
• Stratum corneum thickness analysis

PROTOCOL
TESTED PRODUCTS

Hydrosella™ was diluted in water at 1% and topically applied based on 2mg per cm2 and spread
using a small spatula.

BIOLOGICAL MATERIALS

Explants were prepared from an abdominoplasty coming from a 34-year-old woman. The explants
were placed alive in BEM culture medium at 37°C in a humid, 5%-CO2 atmosphere.

METHOD FOR PREVENTIVE EFFECT

To evaluate the preventive action, the product was applied before delipidation. Hydrosella™ was
applied on skin explants on D0, D2 and D3 just before delipidation on D3.

Untreated explants did not receive any treatment except the renewal of half the culture medium
on D3 and were not delipidated
Delipidated explants did not receive any product application (Hydrosella™).
The explants, except Untreated, were delipidated by 3 applications of 30 µL of an ether/acetone
solution (1:1) on paper disks at the explant surface, for 1 hour each.

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METHOD FOR CURATIVE EFFECT

Curative effect of Hydrosella™ was evaluated, with a single application just after skin delipidation:

Explants were cultivated 3 days before delipidation stress.

Untreated explants did not receive any treatment except the renewal of half the culture medium
on D0 and were not delipidated.
Delipidated explants did not receive any product application (Hydrosella™).
All skin explant (Untreated, Control and Hydrosella™) had the renewal of half the culture medium
on D0.
The explants (Control and Hydrosella™) were delipidated by 3 applications of 30 µL of an
ether/acetone solution (1:1) on paper disks at the explant surface, for 1 hour each.

EVALUATION

Cellular viability analysis

The cellular viability of epidermal and dermal structures was analyzed after staining of formalin-
fixed paraffin-embedded (FFPE) sections according to Masson’s trichrome, Goldner variant.
The staining was assessed by microscopical observation.

Occludin immunostaining
Occludin immunostaining was carried out on FFPE skin sections using a monoclonal anti-occludin
antibody, incubated overnight at room temperature, enhanced with a streptavidin/biotin system
and revealed using VIP, a violet substrate of peroxidase.
The immunostaining was performed manually and assessed by microscopical observation.

Neutral lipids staining

Neutral lipids were stained on frozen sections using the LipidTox™ probe.
Frozen skin sections were fixed, post-chromatized and incubated with LipidTox™, diluted at 1:3000
in PBS, for 45 minutes at room temperature. Nuclei were counterstained with DAPI diluted at a
concentration of 0,1 μg/mL in PBS.
The staining was performed manually and assessed by microscopical observation.

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TauT transporters immunolabelling
TauT transporters immunostaining was carried out on skin sections using a monoclonal anti-TauT
antibody diluted at 1/100 in PBS – 1% BSA and revealed with an Alexa 488 secondary antibody
(green labelling). The immunostaining was performed manually and assessed by microscopical
observation.

Stratum corneum morphology

The SC morphology was assessed by microscopical observation, following both occluding
immunostaining and neutral lipid staining.

Image analysis
Quantification was done on neutral lipid staining (fluorochrome) and TauT labelling.
Image processing was performed using Fiji software29. We used a DAPI dye (blue channel) to
identify nuclei in the epidermis region. The green channel was used to quantify fluorescence
labelling as a result of the LipidTox™ dye in the Stratum Corneum region (SC), or the TauT content
in the epidermis region.
Mean LipidTox™ intensity was computed as a ratio between SC total intensity and area size (SC
mean intensity).
Mean TauT intensity was then computed as a ratio between epidermis total intensity and area
size.
The results are reported as percentage of total fluorescence intensity compared between the
control and the treated assay. Obtained data and percentage variations were submitted to two-
way test t of Student for paired data. The statistical significance value is p<0.05.

RESULTS

After treatment with ether/acetone, lipid protection in the stratum corneum falls sharply, with a
slight recovery after 48 hours.

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1- HYDROSELLA™ SKIN NEUTRAL LIPID PREVENTIVE PROTECTION
ACTION
NEUTRAL LIPID STAINING

D3 Untreated

D3 Delipidated D5 Delipidated

D3 Delipidated + Hydrosella™ 1% D5 Delipidated + Hydrosella™ 1%

We can see that in the delipidated control; the lipids staining (green label) is less intense.

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When the explants are treated before delipidation, Hydrosella™ at 1% helps to maintain the level
of lipids and boost the recovery of lipid protection after 48 hours, protecting the skin's barrier
function.
It has been shown that osmolyte uptake has a role in enhancing the lipid composition of the
stratum corneum, either by stabilizing ABC transporter proteins or by increasing lipids synthesis
in keratinocytes24. This may explain the action mechanism of Hydrosella™ on the lipid layer.

CONCLUSION

Hydrosella™ provides a preventive action for skin lipid protection and increases its
production

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2- HYDROSELLA™ SKIN LIPID CURATIVE PROTECTION ACTION
OCCLUDIN IMMUNOSTAINING

The immunostaining of Occludin appears in violet color on the pictures below.

Untreated T3h control T3h + Hydrosella™ 1%

The decrease in purple color 3 hours after delipidation (occluding immunolabelling) attests to the
reduction in occluding expression and barrier function.
When treated with Hydrosella™ at 1% just after delipidation, the occluding immunostaining is
higher than the delipidated explant, demonstrating a protective effect by Hydrosella™ on barrier
function.

NEUTRAL LIPIDS STAINING

When applied after delipidation, Hydrosella™ at 1% helps to maintain the level of lipids and
accelerate its production, protecting the skin's barrier function.

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T3h Delipidated
T3h Delipidated + Hydrosella™ 1%

D1 Delipidated
Untreated D1 Delipidated + Hydrosella™ 1%

D2 Delipidated
D2 Delipidated + Hydrosella™ 1%

TAUT TRANSPORTERS IMMUNOLABELLING

The secondary antibody contains a green fluorophore. The increase of the green color is correlated
to the increase of TauT transporter.

T3h Delipidated
T0 T3h Delipidated +Hydrosella™ 1%

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TauT transporter increases after application of Hydrosella™, to reinforce the defense of the cells
following delipidation stress. All pictures are at 40x zoom.

STRATUM CORNEUM THICKNESS

Stratum corneum morphology is characterized by two main aspects: lamination and thickness.
Lamination is evaluated based on the empty spaces between corneocytes, also called basket-wave
pattern.

In a well-hydrated stratum corneum, serine protease activity is optimal, leading to

corneodesmosomes destruction. Corneocytes remain linked only by corneodesmosomes
preserved by tight junctions, leaving empty spaces showing the laminated aspect31,32. This aspect
is associated with well-moisturized skin.
In a poorly-hydrated stratum corneum, serine proteases activity is reduced, leading to a compact
stratum corneum. This aspect is associated with dry skin.

In the pictures, an increase in the stratum corneum thickness is observed after treatment with
Hydrosella™ compared to the delipidated control:

T3h Delipidated T3h Delipidated+ Hydrosella™ 1%

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The area of the stratum corneum was also measured.

We observed that the area of stratum corneum significantly decreases after delipidation.
Hydrosella™ helps to keep a thicker SC with a laminated aspect. This is clearly representative of a
well-hydrated skin.

CONCLUSION

Hydrosella™ increases skin neutral lipid production following dehydration to prevent

further water loss

Hydrosella™ enhances the stratum corneum thickness to maintain a well-hydrated skin

Hydrosella™ increases tight junction proteins expression (occludin) to restore and to

improve skin barrier function.

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CLINICAL STUDIES

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CLINICAL EVALUATION OF HYDROSELLA™ ON DRY

SKIN: FAST AND LONG-LASTING ACTION

OBJECTIVE
The aim of the study was to assess the moisturizing effect of Hydrosella™ at 1% in a simple gel,
after a single application (short term study), versus placebo.

PROTOCOL
SUBJECTS

This study was carried out on 10 healthy female subjects with normal to dry skin aged from 24 to
62 (mean 43.1).

TEST CONDITIONS

The evaluations were carried out on the volar surface of the forearms at baseline (T0) and after 1
(T1h), 24 (T24h), 48 (T48h) and 72 hours (T72h) from product single application in comparison
with an untreated area and a placebo-treated area. Products were applied on right and left
forearms according to a randomization scheme.
Measurements were performed by means of a non-invasive bioengineering technique able to
quantify skin moisturizing (CORNEOMETER® CM825, Courage+Khazaka Electronic GmbH) and
trans epidermal water loss (TEWL) (TEWAMETER TM300®, Courage+Khazaka Electronic GmbH).

TESTED CREAMS

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METHODS
SKIN MOISTURIZING

The skin moisturizing measurement was made using the internationally recognized
CORNEOMETER® instrument. This measurement is based on the completely different dielectric
constant of water (81) and other substances (mostly < 7).

TRANS EPIDERMAL WATER LOSS (TEWL)

Measurement of trans-epidermal water loss was made using the internationally recognized
TEWAMETER® instrument.

STATISTICAL METHODS
For each product, the data obtained at different experimental times were submitted to a bilateral
Student’s t test for paired data vs T0. Moreover, at each experimental time, each product was
compared to the others and to the untreated area through a Student's t test for paired data. The
statistical significance value is p<0.05 (**p<0.01, ***p<0.001).

RESULTS
Hydrosella™ rapidly improves skin moisture after 1 hour with a sustainable effect lasting at least
72 hours after product application.

Up to
+29%

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Up to
-11%

Following application of Hydrosella™, the TEWL instantly decreased and the reduction lasted for
72 hours, showing the efficacy of the product in supporting the improvement of skin barrier
function. Moreover, this reduction was highly significant vs the untreated and placebo area at
each time

CONCLUSION

Hydrosella™ rapidly increases skin moisture and reduces water loss values with a
sustainable effect over time up to 72hrs after single application.

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CLINICAL EVALUATION OF HYDROSELLA™ ON THE

AGRESSED SKIN OF ACTIVE PEOPLE
INTRODUCTION
Swimming is the most challenging sport for the skin and hair. Skin has a protective layer of lipids
that lubricate it and make it semi-waterproof. Water can partially remove the skin lipid layer. This
situation is worsened for swimmers who are exposed to chlorine and other pool chemicals, as they
accelerate the removal of lipids from the skin. Once skin lipids are removed, skin loses its first
protective layer, making it feel tight and itchy. The situation is the same for active people training
on a regular basis who tend to take several showers a day. The repeated contact with soap and
other surfactants will also remove the skin's protective lipid barrier and can cause skin irritation,
dryness and scaling.

OBJECTIVE
The aim of the study was to assess the moisturizing effect of Hydrosella™ on the skin of active
people after the aggressive effects of chloride water, repetitive showers, etc.

Two studies were performed on 2 different panels.

1- Short-term study following a single application on active men and women with dry skin;
2- Long -term study for 28 days, application twice daily, on active men only.

PROTOCOL
SHORT TERM STUDY SUBJECTS

8 subjects for Hydrosella™, and 8 subjects for placebo were selected, aged from 24 to 45 (mean
31), all with dry skin (corneometry UI<30), both men and women, all in contact with chloride water
several times a week.

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Corneo Corneo
Sex Sport Frequency
placebo arm active arm
M 29 swimming + handball 2 x swimming + 3 x handball
M 27 28 Triathlon 7 times
F 25 28 Aquagym 3 times
F 25 26 swimming 3 times
M 23 23 swimming + tennis 3 x swimming + 2 x tennis
F 22 21 swimming 3 times
F 22 21 swimming + yoga 2 x swimming + 1 x yoga
M 20 25 swimming 5 times
M 23 swimming + tennis 3 x swimming + 2 x tennis

SUBJECTS LONG TERM STUDY

10 men were selected, all of them in contact with chloride water several times a week.

Age Sport Frequency

26 swimming + tennis 3 x swimming + 2 x tennis
22 Triathlon 7 times
41 swimming 5 times
24 swimming + tennis 3 x swimming + 2 x tennis
32 swimming + handball 2 x swimming + 3 x handball
25 Triathlon 4 times
24 swimming + running 2 x swimming + 2 x running
30 Triathlon 7 times
38 swimming 5 times
33 Waterpolo + body building 4 x waterpolo + 3 x body building

TEST CONDITIONS

2 formulas were evaluated in a double-blind study, one containing Hydrosella™ at 2%, the other
a placebo. They were randomly applied on one arm of each subject (one formula/arm).

Short-term study: One application of product or placebo followed by measurement after 1 hour
and 24 hours. During this time, the subject should avoid any physical activity and not shower.

Long-term study: Formulas were applied twice a day for 28 days. Measurements were made
before the first application, after 14 days and at the end of the study.

***The test took place in the south-east of France in June 2019, during a heatwave (outside
temperature above 40°C). TEWL reading at 28 days showed aberrant values. Considering that the

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human skin plays a role in regulation of the body temperature (by sweating) it is believed those
aberrant values are explained by the extreme weather during the clinical trial. Therefore, no data
were available at 28 days for the TEWL***

TESTED CREAMS

METHODS
SKIN MOISTURIZING

The skin moisturizing measurement was made using the internationally recognized
CORNEOMETER® instrument. This measurement is based on the completely different dielectric
constant of water (81) and other substances (mostly < 7).

TRANS EPIDERMAL WATER LOSS (TEWL)

The measurement of the trans epidermal water loss was made using the internationally
recognized TEWAMETER® instrument. The instrument used was a Tewameter 300®
(Courage+Khazaka, Electronic GmbH).

STATISTICAL METHODS
For each product, the obtained data at the different experimental times were submitted to
bilateral Student’s t-test for paired data vs T0. Moreover, at each experimental time, each product
was compared to the others and to the untreated area though Student’s t-test for paired data.
The statistical significance value is p<0.05 (*p<0.05).

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RESULTS
SHORT-TERM STUDY:

Amongst the subjects with dry skin area (corneometry value <30, 8 people treated with
Hydrosella™ and 8 treated with the placebo), a significant improvement of the corneometry value
of 27.5% after 1 hour and 17.8% after 24 hours was noticed vs T0 for volunteers treated with
Hydrosella™ at 2%.

Up to
+56%
Up to
+54%

An increase in water content was observed for 100% of the subjects with dry skin after 1 hour and
75% after 24 hours.

LONG-TERM STUDY:

On active men taking 2 showers a day and practicing sports several times a week, skin hydration
improved after 14 and 28 days with a twice-daily application of Hydrosella™ 2%. Corneometry and
TEWL showed significative improvement.

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Up to
+ 190
%

Significant water content improvement was observed for 100% of the men after 14 days and 89%
after 28 days.

Up to
-60%

Trans epidermal water loss decreased after 14 days with Hydrosella™ at 2%, showing an
improvement in barrier function efficacy, even in harsh conditions (swimming pools, sweating,
showers, etc.).

CONCLUSION

Hydrosella™ enhances skin hydration and reduces water loss rapidly and with a
sustainable action on active people submitting their skin to extreme conditions,

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CLINICAL EVALUATION OF HYDROSELLA™

MOISTURIZING EFFECT IN RINSE-OFF APPLICATION
INTRODUCTION
Adequate hydration is crucial to achieve and maintain healthy skin. When the skin is dehydrated,
it can lose its radiance, become rough, and lose its elasticity. Rinse-off products, such as cleansers
and shampoos, can potentially be dehydrating to the skin. This is because they often contain
surfactants that can strip away the skin’s natural oils and disrupt its moisture balance. Frequent
use of harsh rinse-off products can lead to dryness, tightness and irritation. Additionally, the use
of hot water during rising can further contribute to moisture loss.

OBJECTIVE
The aim of the study was to assess the moisturizing effect of Hydrosella™ at 2% in a soap bar, after
a single application, versus placebo.

PROTOCOL
SUBJECTS

This study was carried out on 20 healthy female volunteers aged from 34 to 69 years old (mean
54.5) with dry skin on legs (Moisturizing index by corneometry <50).

TEST CONDITIONS

Delimitation of six skin areas were made on subject’s legs. Three areas with a comparable
moisturizing index were selected for the corneometric measurements. Three other areas were
selected for the Dermatoscope C-Cube acquisitions. The evaluations were carried out at baseline
(T0) and after 1 (T1h) and 4 (T4h) hours from product single application in comparison with an
untreated area and a placebo-treated area. Products were applied on selected areas according to
a randomization scheme.

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TESTED BAR SOAP

METHODS
SKIN MOISTURIZING

The skin moisturizing measurement was made using the internationally recognized
CORNEOMETER® instrument. This measurement is based on the completely different dielectric
constant of water (81) and other substances (mostly < 7). During the trial, each measure was
repeated three times and the mean value was recorded.

ACQUISITIONS WITH DERMATOSCOPE C-CUBE®

Skin images were acquired with the C-Cube® Dermatoscope device (PIXIENCE) which produce high
resolution skin images (10 million pixels 2D capture (UHD)). The captures were taken on 3 areas:
the treated and the control areas.

STATISTICAL METHODS
For each product, the obtained data at the different experimental times were submitted to two-
way Student’s t-test for paired data vs T0. The statistical significance value is p<0.05 (*p<0.05,
**p<0.01).

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RESULTS

In just one rinse-off application, HydrosellaTM significantly boosts skin water content, providing
rapid moisturization to the skin. Indeed, an increase in water content was observed for 100% of
the subjects after 1 hour and 67% after 4 hours.

Use of HydrosellaTM in rinse-off application leads to an improved skin appearance, so the skin
looks smoother and less dry.

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CONCLUSION

Hydrosella™ rapidly increases skin hydration and enhances overall skin appearance when
used in rinse-off conditions

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CONCLUSION
Hydrosella™ is a new generation natural hydration/moisturizing active ingredient. Using the
organic osmolytes strategy, Hydrosella™ stimulates the production of organic osmolyte
transporters and organic osmolyte synthesis to accelerate keratinocyte volume recovery.
Simultaneously, Hydrosella™ restores the skin's barrier function by increasing lipid production,
stimulating tight junction proteins and increasing the thickness of the stratum corneum. This
multi-target moisturizing active ingredient acts on the root cause of skin dryness, providing a rapid
and continuous improvement of skin hydration and skin's water loss both in rinse-off and leave-
on applications.

Hydrosella™ is an extract from a unique variety of hibiscus (Hibiscus sabdariffa L.), called Wild
Rosella, that has now been naturalized in the tropical regions of Australia, where drought periods
are common. This organically grown variety has been specially selected for its high natural betaine,
content, an organic osmolyte. Standardized in naturally-occurring betaine from Wild Rosella,
Hydrosella™ guarantees a high clinical efficiency and stable action from batch to batch. Our
unique sustainability policy, combined with a rigorous supply chain, provides full traceability from
plantation to jar. Being China-compliant, Hydrosella™ offers a new way to hydrate and moisturize
skin.

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COSMETIC APPLICATIONS
• Moisturizing and Hydrating skin care
• Skin repair treatment
• Daily skin care and protective care
• Face and Body skin care
• Spa treatments
• Men’s skin care
• Athleisure care
• Rinse-off applications

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RECOMMENDED USE
DESCRIPTION
Hydrosella™ is a hydro dispersible active ingredient for hydration of the skin. It is in the form of a
pink-red liquid with a characteristic odour.

FORMULATION WITH Hydrosella™

1. LEVEL OF USE

The recommended dosage of Hydrosella™ is up to 2%.

When Hydrosella™ is added to a formula, it leads to a very light color in gels and gel-creams
(shades of pink) and a non-significant impact in emulsified formulas.
2. PROCESS OF USE

Hydrosella™ must be preferably introduced at the end of the formulation process, below 40°C.
However, Hydrosella™ is stable when heated up to 6 hours at 80°C.
3. PH IMPACT

pH value must be adjusted between 4.0 and 8.0 to ensure proper organoleptic characteristics of
the formulated product.
Outside the recommended pH range, the stability and the aspect of the formula should be
evaluated case by case by the formulator.
Note that at acid pH, formulas with Hydrosella™ will have a pinker color while at basic pH, they
will be more yellow.

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ADDITIONAL DATA

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EFFECT OF HYDROSELLA™ ON INFLAMMATION IN

NORMAL HUMAN DERMIS FIBROBLASTS
INTRODUCTION
When stimulated with the inflammatory cytokine IL-1α, cultured human dermal fibroblast (NHDF)
responds by secreting IL-8. IL-8 mediates inflammatory reactions in the skin through the and other
immune cells to invade injured or inflamed tissue. It has been shown that organic osmolytes like
betaine decreases inflammatory mediators’ secretion. By acting on organic osmolyte content,
Hydrosella™ may induce a decrease in inflammation33.

OBJECTIVE
Dry skin is often linked to inflammation. The aim of this study was to evaluate Hydrosella™,
betaine and erythritol effects on the reduction of inflammation in normal human dermal
fibroblasts (NHDF).

PROTOCOL
TESTED PRODUCTS

Hydrosella™ was tested at the following concentrations: 0.05%; 0.1%; 0.2% and 0.4%.
Betaine was tested at the following concentrations: 2.5; 5; 10 and 20µM.
Erythritol was tested at the following concentrations: 0.005%; 0.01%; 0.02% and 0.04%.
IL-1α was used as activating factor, at 0.0003 ng/mL.
Hydrocortisone was used as positive control at a final concentration of 0.05 µg/mL.

BIOLOGICAL MATERIALS

NHDF are fibroblasts isolated from human dermis and maintained in a specific medium composed
of DMEM containing 10% serum, 1% antibiotics and 1% L-glutamine at 37 °C under 5% CO2 and
95% humidity.

METHOD

NHDF were seeded at the concentration of 2.104 cells/well. After 1 day, medium with serum was
removed and cells were treated with IL1-α, as an inducer of IL-8 production, in the
presence/absence of Hydrosella™, betaine, erythritol or positive control.

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EVALUATION

The supernatants were collected and IL-8 released from fibroblasts was determined by enzyme-
linked immunosorbent assay (ELISA).

STATISTICAL ANALYSIS

Obtained data and percentage variations were submitted to two-way test t of Student for paired
data. The statistical significance value is p<0.05 (*p<0.05, **p<0.01, ***p<0.001).

RESULTS

Hydrosella™ has a clear anti-inflammatory activity, decreasing the pro-inflammatory cytokine IL-
8. Betaine seems in part responsible for the result, and erythritol has no activity.

CONCLUSION

Hydrosella™ decreases the inflammation of the dermis

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