Effipulp - RAHN-Group

Effipulp®

Anti-aging, Plumping & Detoxifying

2 Cosmetic Active Ingredients

Ppt Effipulp® ENG

January 2012

1. Presentation of Effipulp®

2. The issue of aging

3. Biological activity

• Restoring natural defenses and detoxification systems

• Moisture redensification & strengthening of the stratum corneum

4. Clinical study: Plumping effect

Table of contents


Ppt Effipulp® ENG

January 2012

• Avocado (Persea Gratissima), mainly Fuerte and Haas varieties, collected in Africa and Latin America

• Family: Lauraceae (laurels), subfamily: Persea (ex. bay laurel, cinnamon, camphor)

• Climacteric fruit, ripening when the fruit is picked

• Native to Mexico, consumed 10,000 years ago by the Aztecs and Mayas, then imported to Europe in the 17th century

Avocado: presentation & history


Ppt Effipulp® ENG

January 2012

Avocado: Sourcing and Use based on CSR

• Laboratoires Expanscience is confirmed as Trading member of UEBT* since end Dec. 2011

*Union for Ethical BioTrade (http://www.ethicalbiotrade.org/)

• Exemple: top-grafting of old Avocado trees with young ones:

- work on existing cultivated lands (avoid deforestation)

- get back younger trees, able to provide fruits

• Total exploitation of Avocado fruit

http://www.ethicalbiotrade.org/


Ppt Effipulp® ENG

January 2012

Avocado: a total exploitation of the fruit…

Pressure

Crude Oil

Molecular Distillation

Unsaponifiables pharmaceutical application (API)

Sliding & drying

Avocado Oil HAREF

Avocado cakes

Esterification

5 a Avocuta®

Hydrolysis

Effipulp®

Avocado powder Fresh fruit

composition:

– 70% water

– 21% lipids

– 4% glucides

– 2% proteins

– 2% fibers

– 1% ashes

Refining


Ppt Effipulp® ENG

January 2012

… and benefits of a pharmaceutical supply

• Specific preparation of Avocados (included in a patent):

1. water eliminated through drying under controlled conditions

2. oil obtained by pressure

• Unique quality of Avocado “cakes”:

- preserved thanks to the preparation method

- concentrated in proteins

• Laboratoires Expanscience unique supplier of Avocado peptides.


Ppt Effipulp® ENG

January 2012

Green process without organic solvent

> Enzymatic hydrolisis of delipidated pressed Avocados (Cellulase – Protease) to obtain peptides

> Purification through ultrafiltration (15 kDa) and nanofiltration (1 kDa)

> Bleaching

> Addition of organic maltodextrin (50%) & freeze-drying

Process respecting the naturalness of the fruit


Ppt Effipulp® ENG

January 2012

• No residual proteins (> 10 kDa)

• Reproductibility of the composition in peptides (distribution in molecular weight)

HPTLC chromatography

Characteristics Beige powder with characteristic odour

Protide content (Kjeldahl method) ≥ 15%

Amino a nitrogen content (UV spectrophotometry)

4.0-8.0 g/100 g

Maltodextrin 45.0-55.0 g/100 g

Total micro-organism count

Yeast and mould count

Pathogens

<100 CFU/g

<100 CFU/g

None

Analytical specifications


Ppt Effipulp® ENG

January 2012

• Shelf life: 3 years

• Packaging: 100 g and 1 kg

• INCI name: Hydrolyzed Avocado Protein and Maltodextrin

• CAS numbers: 1085861-52-4 ; 9050-36-6

• REACH status: Pre-registered

• Patent ref.: WO 2005/105123 - WO 2010/052312

Technical and regulatory data / patents

The issue of aging &

consequences for the skin


Ppt Effipulp® ENG

January 2012

Production of Reactive Oxygen Species (ROS)

• Endogenous: cellular metabolism (cellular respiration)

• Exogenous: UV radiation, stress, pollution etc.

Antioxidant defenses

• Enzymatic: SOD, catalase, glutathion peroxidase

• General: Vit. E, vit. C, vit. A

Systems repairing and eliminating oxidized protein

• Repair: thioredoxin, glutaredoxin etc.

• Elimination (cytosolic): Proteasome

Main factors of skin aging


Ppt Effipulp® ENG

January 2012

Young skin: Homeostasis between ROS production and natural defenses

Natural defenses

Endogenous and exogenous

production

ROS

ROS

Antioxidant

Repair and elimination systems

Antioxidant

Repair and elimination systems



Ppt Effipulp® ENG

January 2012

Natural defenses

Endogenous and exogenous

production

Mature, weakened skin: Imbalance in homeostasis, cellular damage



Ppt Effipulp® ENG

January 2012

Cellular functions:

• Proliferation and differentiation of keratinocytes and fibroblasts

• Syntheses (HA, collagens, elastin,…)

• Protection systems (proteasome, thioredoxin,…)

ROS

Oxidation

PROTEINS

DNA mutation

Peroxidation

LIPIDS

Str

ess

ROS consequences and skin aging

CONSEQUENCES

Epidermis: alteration of the protective barrier and therefore of the moisture balance

=> dehydrated and stressed skin: loss of moisture, volume

Dermis: alteration of the extra-cellular matrix => mature, weakened skin:

loss of suppleness, firmness and radiance

Biological activity: restoration of the natural

defenses and detoxification systems


Ppt Effipulp® ENG

January 2012

• Enzymatic systems repairing oxidized protein (reduction): • Thioredoxin / Thioredoxin Reductase

• Glutaredoxin

• Methionine Sulfoxide Reductases

• Intracellular proteolytic deterioration The proteasome, potential recycling of amino acids

Sub-units Catalytic activities

Systems repairing and eliminating oxidized protein


Ppt Effipulp® ENG

January 2012

• Human dermal fibroblasts, treated with 0.005% Avocado peptides over 48 h

• Gene expression analysis by RT-PCR

Activity on thioredoxin - repair system

0,005 %

Avocado peptides

0,01% Effipulp®

*** p<0.001

relative quantity of the gene expression

(normalized)

0 ,0 0

0 ,5 0

1 ,0 0

1 ,5 0

2 ,0 0

2 ,5 0

3 ,0 0

C o n t r o l A v o c a d o p e p t i d e s

0 , 0 0 5 % A I

Qu

an

tit

é R

ela

tiv

e n

or

ma

lis

ée

p

ar

Y

WH

AZ + 1 7 7 %

* * *

0 ,0 0

0 ,5 0

1 ,0 0

1 ,5 0

2 ,0 0

2 ,5 0

3 ,0 0

C o n t r o l A v o c a d o p e p t i d e s

0 , 0 0 5 % A I

Qu

an

tit

é R

ela

tiv

e n

or

ma

lis

ée

p

ar

Y

WH

AZ + 1 7 7 %

* * *

non-treated cells Avocado peptides 0,005%

Effipulp® stimulates the gene expression of thioredoxin


Ppt Effipulp® ENG

January 2012

• “Senescent” fibroblasts (successive replications) treated over 1 hour and 45 minutes with 0.005% Avocado peptides, or by PA28 (proteasome activator)

• Measurement of enzymatic activity of proteasome (luminescent substrate)

Senescence slows the proteasome’s activity as well as the gene expression

of the sub-unit1

Effipulp® restores the proteasome’s activity as well as the gene expression

of the sub-unit1

Activity on the Proteasome

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

1 6 0

" Y o u n g c e l l s " " S e n e c e n t

c e l l s "

R é f é re n c e

( P A 2 8 a c t i v a t o r

4 n g / m l )

A P 0 , 0 0 5 % A I

Ac

tiv

ité

(%

du

té

mo

in)

+38%

° ***

+43%

°

-32%

° vs senescents cells

*** p<0.001

pro

teasom

e a

ctivity (

%)

"Young cells" "Senescent cells"

Ref. PA28 (4 ng/mL)

Avocado peptides 0,005%


Ppt Effipulp® ENG

January 2012

• “Senescent” fibroblasts (successive replications) treated over 72 hours with 0.005% Avocado peptides, or with BHA at 150 µM

• Measurement of the quantity of oxidized proteins (in fibroblasts) using a specific fluorescent probe

The rate of protein oxidation increases

with senescence

Effipulp® inhibits the accumulation

of oxidized proteins

Activity on oxidized proteins

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

"Y o un g ce l l s " "Se n e s ce n t

ce l l s "

R e fe re nce

(B H A 1 5 0 µM )

A P 0 ,0 0 5% A I* p<0.05

-57%

° *

+35%

-44%

° *

Oxydiz

ed p

rote

ins

(flu

ore

scence inte

nsity)

° vs senescents cells

"Young cells" "Senescent cells"

Ref. BHA (150 µM)



Ppt Effipulp® ENG

January 2012

Protective effect against lipid peroxidation

P=50%

P=64%

Quantitity of lipidic peroxydes

Effipulp® modulates lipid peroxidation and therefore protects the integrity of cellular membranes

• Jurkat cells (cellular model) subject to H2O2 stress and treated with 0.05% Avocado peptides, or with BHA (anti-oxidant ref.) over 30 minutes

• Measurement of the peroxidation of membrane lipids using a specific fluorescent probe (C11-fluor, flow cytometry)


Ppt Effipulp® ENG

January 2012

Restoration of cellular functions for:

Hygroscopic molecules and proteins syntheses

Cohesion of the stratum corneum

Restoring natural defenses & detoxification systems

Protective effect of Effipulp®

oxidized proteins

Antioxidant effect of Effipulp®

peroxidized lipids

• Repair by thioredoxin

• Elimination by proteasome Cellular membranes protection

Biological activity: moisture redensification & strengthening of the

statum corneum


Ppt Effipulp® ENG

January 2012

• Moisture content with age

GAGs and in particular in hyaluronic acid guaranty

a natural hydration of the dermis and epidermis, returns density, elasticity and comfort (trapping of water, up to 1,000 times its weight)

=> Aging: GAGs HA = significant reduction in the skin’s volume (by age 50, half of the HA has disappeared)

• Stratum corneum

The cohesion of the SC limits imperceptible water loss and therefore maintains the moisture level, in particular in the epidermis.

=> Aging: disruption of SC

Hyaluronic acid

Water

Aging: moisture balance and maintenance


Ppt Effipulp® ENG

January 2012

Epidermal moisture redensification

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

C o n t r o l R e t o in o ic a c id

0 , 1 µ M

A v o c a d o

P e p t id e s

0 , 0 0 5 % ( A I )

* p<0.05 ** p<0.01

+146%

+31%

*

**

HA q

uantity

(n

g/m

l)


Non treated cells Ref. Retinoic acid (0,1 µM)

Effipulp® stimulates the release of Hyaluronic Acid by keratinocytes

• Normal human keratinocytes treated over 72 hours with 0.005% Avocado peptides, or with retinoic acid at 0.1 µM (ref.)

• Measurement of the release of Hyaluronic Acid (ELISA) in cell culture supernatants


Ppt Effipulp® ENG

January 2012

Dermal moisture redensification

0

0 ,5

1

1 ,5

2

2 ,5

3

C o n t ro l "S e n e s c e n t C e lls " A v o c a d o p e p t id e s

0 ,0 5 % ( A I)

*** p<0.001 ** p<0.01

HA q

uantity

(µ

g/m

l)

-64%

+91% vs senescent cells

**

***

"Young cells" "Senescent cells" Avocado peptides 0,05%

• Normal human fibroblasts “senescent” aged with H2O2 treated over 8 days with 0.05% Avocado peptides,

• Measurement of the release of Hyaluronic Acid (ELISA) in cell culture supernatants

Effipulp® stimulates the release of Hyaluronic Acid by fibroblasts under senescent conditions


Ppt Effipulp® ENG

January 2012

• Topical application of an emulsion with 0.6% Effipulp® vs. placebo on normal skin explants over 72 hours

• Immunostaining of Hyaluronic Acid (green) & image analysis quantification

Effipulp® stimulates the synthesis of Hyaluronic Acid

Epidermis

Dermis

Skin moisture redensification: ex-vivo confirmation


Ppt Effipulp® ENG

January 2012

• Topical application of an emulsion with Effipulp® (0.6%) vs. placebo on delipidated skin explants (model for dry skin) over 3 hours

• Marking of Filaggrine (SC cohesion, precursor of NMF) and Transglutaminase (corneal envelope) by immunofluorescence (green)

Placebo Effipulp® 0.6% Placebo Effipulp® 0.6%

Effipulp® stimulates the synthesis of molecules involved in the cohesion & formation of the stratum corneum

Corneal layer strengthening: epidermal cohesion

Filaggrine (FG) immunostaining Transglutaminase (TG) immunostaining


Ppt Effipulp® ENG

January 2012

• Synthesis of hygroscopic molecules

• Hyaluronic acid in the epidermis and dermis

• GAGs (+34%, in-vitro, keratynocytes, 0.005% AI)

Moisture redensification

• Strengthening of the corneal layer

• Filaggrine, transglutaminase

• Ceramides (+30%), cholesterol (+12%)

(reconstructed epidermis, 0,005% AI)

Preservation of moisture content

Redensification & preservation of moisture content


Ppt Effipulp® ENG

January 2012

Mechanism of action


Ppt Effipulp® ENG

January 2012

Mechanism of action

Clinical study: Plumping effect of

Effipulp®

Optical Coherence Tomography (OCT)


Ppt Effipulp® ENG

January 2012

Stratum Corneum

Granulosum

Spinosum

Dermis

Epidermis

• Innovative and precise technique

> Specific optical measurement for the epidermis (infrared laser, very high definition images)

> 2 acquisition of 400 joint pictures over a distance of 1,6 mm on the same area

> Epidermis thickness and volume quantification (SC excluded)

OCT technique / data by age range

Neerken, S. et al, Journal of Biomedical Optics, 2004

• Bibliographical study comparing the thinning of the epidermis during aging between 2 groups (15 pers. each)

8 0 %

8 5 %

9 0 %

9 5 %

1 0 0 %

Y o u n g

( 2 2 , 5 y e a r s )

A g e d

( 5 5 , 3 y e a r s )

Epidermis thickness evolution by aging

-8.4%

(22,5 years) (55,3 years)


Ppt Effipulp® ENG

January 2012

• Comparative study, double-blind vs. placebo

• Effipulp® tested at 0.6% (face cream)

• 49 women - 2 groups of 25 subjects

Thickness values for both groups are comparable with T0

• Inclusion criteria:

age 40-56, average age: 49

dry and sensitive skin types (stinging test)

• Term: 56 days – 2 applications/day on the face

• Measurement (cheeks) of the variation in thickness in relation to the area, or the volume of the epidermis

Clinical study: protocol


Ppt Effipulp® ENG

January 2012

> Placebo: no significant difference between D0 and D56 (+1,9%)

> Cream with Effipulp®: +6.1% significant increase (average) in epidermis volume after 56 days

Results


Ppt Effipulp® ENG

January 2012

Results: 3D visualisation

Pores or hair-sebaceous structure

Epidermis thickness

Skin surface (below SC)


Ppt Effipulp® ENG

January 2012

Activity summary

Protective and

antioxidant effect

• Oxidized proteins - Thioredoxin and Proteasome

• Peroxidized lipids for cellular membranes integrity

Restoration of cellular

functions

•Synthesis of Hyaluronic Acid - Dermis & Epidermis

•Protein synthesis - Cohesion of stratum corneum

Plumping and redensifying

effect

•Increase in the volume of the epidermis

•Strengthening of the stratum corneum for moisture preservation


Ppt Effipulp® ENG

January 2012

• ANTI-AGING PLUMPING AND DETOXIFYING

• Strengthening the natural defense and detoxification systems - the Proteasome - to restore cellular functions

• Plumping the epidermis and dermis by stimulating the synthesis of hyaluronic acid

• Strengthening the stratum corneum to preserve moisture

• Applications: Face, lips, chest and hands

• Restores lost volume to rejuvenate skin

• Returns firmness and elasticity to the skin

• From the first signs of aging to mature, weakened, dehydrated and stressed skin

• Can complement esthetic cosmetic procedures

• Recommended dose: 0.1% to 0.6%

Conclusion


Ppt Effipulp® ENG

January 2012

To find out more…

Laboratoires Expanscience

10, Av de l’Arche

92419 COURBEVOIE CEDEX

www.expanscience-ingredients.com

Maud CHAPIS François MONTCRIOL

Tel: +33 1 43 34 75 39 +33 1 43 34 75 81

Fax: +33 1 43 34 61 19 +33 1 43 34 61 19

[email protected] [email protected]

http://www.expanscience-ingredients.com/



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Effipulp - RAHN-Group