AN ACTIVE SHELTER TO PROTECT CELLSPROTECT CELLS

v5

WHERE IS THE MARKET GOING TO?

Niche segments, such as teenagers and men, are growing in the skin caremarket.

They are also following the trends settled by women’s skin care.

PREVENTION is a rising worry.

Teenagers

beginning preventive care are

Men

More advanced grooming products:…beginning preventive care arein favor of prevention ratherthan waiting until it’s too late!

More advanced grooming products:mass and premium brands areoffering more specific products.

Both groups are becoming more interested in retaining a youthful appearance

PREVENTIVE COSMETICS is about convincing the society of what science already knows: that acting on your skin TODAY will minimise its damage TOMORROWg y g

WHERE IS THE MARKET GOING TO?

Big players are more concerned withprevention and how important it is.

RADICAL SCAVENGING – CLAIM OR REALITY?

Healthy skin is rich in an intrinsic antioxidative defense system

IN THE SKININ THE SKIN

Healthy skin is rich in an intrinsic antioxidative defense system.

In old skin or under oxidative stress, this defensesystem becomes more and more deficientsystem becomes more and more deficient.

IN THE MARKETIN THE MARKET

Skin care products with antioxidative claims are one of the most fast growing marketfor cosmetics worldwide.

BUT 73% f d t th t l i ti id ti… BUT 73% of products that claim antioxidativeproperties showed no or very low antioxidative activity! ** Gematria Test Lab GmbH – AP

method (HPC Today, n 1/2009)

We are finding more and more products in the skin care market

Adding SPF and UVA protection is a major trend in the skin care market, but not theonly one…

g pwith radical scavengers claims:

SPF is necessary but not enough

RADICAL SCAVENGING – CLAIM OR REALITY?

REQUIREMENTS FOR ANTIOXIDANTS AND FORMULATIONS *

High antioxidative capacity and reactivity in order to neutralize the very short time living ROS in High antioxidative capacity and reactivity in order to neutralize the very short time living ROS in the skin.

Antioxidants used in formulations should not be converted into reactive radical species themselves.

Antioxidants should be sufficiently stable inside a cosmetic formulation.

The lipophilic or hydrophilic character of antioxidants determines the distribution inside the skin tissues and makes them more or less accessible to free radicals.tissues and makes them more or less accessible to free radicals.

The cosmetic formulation may enhance or hinder the penetration ability of actives inside the skin.

* Gematria Test Lab GmbH

REACTIVE SPECIES

• Antioxidant is an old, limited concept.

• New research requires NEW TERMINOLOGY:• New research requires… NEW TERMINOLOGY:

ROS Reactive Oxygen Species

R ti Nit g S i

RCS

RNS Reactive Nitrogen Species

Reactive Carbonyl SpeciesRCS Reactive Carbonyl Species

REACTIVE SPECIES

External Factors

RCS

Protein Crosslinking

Lipid Peroxidation

RCS

Protein Crosslinking

DNA damage

ROS

Cellular necrosisRNS

Internal Factors

ROS AND RNS

U d l di i ROS l d f h ll b d

ROS (REACTIVE OXYGEN SPECIES) AND OXIDATIVE STRESS

• Under normal conditions, ROS are cleared from the cell by endogenous antioxidant enzymes.

• Imbalance between the rate of ROS induced oxidative damage and the rate that antioxidants repair the damage and remove ROS is the level of oxidative stress.

• Skin is a major target of oxidative stress due to ROS originated in the environment and in the skin itself, which causes aging signs such as wrinkles, lost of elasticity, and age spots.

• Nitrosative stress occurs when the generation of RNS in a system exceeds the system’s ability to neutralize and eliminate them.

RNS (REACTIVE NITROGEN SPECIES)

the system s ability to neutralize and eliminate them.• Nitrosative stress may lead to nitrosylation reactions (with NO•) that can

alter protein structure thus inhibiting normal function.

PEROXYNITRITE (ONOO-)

NITRIC DIOXIDE (NO2•)

NITRITE (NO2-)

NITRIC OXIDE (NO•)

NITROXYL ANION (NO-)

LIPOCHROMAN-6: VERSATILITY

Li hLi h 6 6 d i i h d bl tid bl tiLipochromanLipochroman--6, 6, due to its two reactive centers, has a double actiondouble action:

ROS scavenger ROS scavenger RNS scavengerRNS scavenger

OCH3O1

27

8

Lipochroman-6

HO3

456

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (I)

1. RNS Capture1. RNS Capture • The nitration of tyrosine residues of proteins is an irreversiblereaction which compromises cyclic interconversion between

RNS

phosphorylated/non-phosphorylated tyrosine, necessary inactivation/deactivation of enzymes and receptors.

• Evaluation of reactivity between tyrosine and peroxynitrite atdifferent concentrations of Lipochroman-6, determined by HPLC.

OH

O

OH

O

O2Nperoxynitrite

NH2HO

NH2HO

Tyrosine 3-Nitrotyrosine

Lipochroman-6 protectsp penzymes from inactivation

Inhibits the reaction between tyrosine and peroxynitrite in a dose-dependent manner.peroxynitrite in a dose dependent manner.

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (II)

2. ROS Capture (I)2. ROS Capture (I) • The ability of Lipochroman-6 to inhibit lipid peroxidationwas evaluated in incubations of sprague dowley rat

ROS

( )

microsomes throug a TBARS test.

TBARS assay

COMPOUND IC50 (µM)

LIPOCHROMAN-6 1.04 ± 0.13

IDEBENONE 11.50 ± 0.65

KINETIN >1000

LIPOIC ACID >1000LIPOIC ACID 1000

Lipochroman-6 proved to inhibit lipid peroxidation atl i d h i ROSlower concentrations, compared to other anti-ROS

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (III)

2. ROS Capture (II)2. ROS Capture (II) • The antioxidant activity of Lipochroman-6 was evaluatedas a part of a study for an interlaboratory comparison to

ROS

( )

assess the antioxidant potential of several compounds1.

Rank Substance Mean

11 LIPOCHROMANLIPOCHROMAN 66 0 380 38

TBA assay (2-Thiobarbituric acid)

Protection of lipid structures11 LIPOCHROMANLIPOCHROMAN--66 --0.380.38

2 BHT -0.06

3 Trolox 0.23was the best of five antioxidants

3 Tocopherol 0.31

3 4-MBC 0.38

in the interlaboratory comparison.

1 J. Buenger et al, International Journal of Cosmetic Science, 2006, 28, 135–146

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (IV)

3. Determination of the 3. Determination of the AntioxidativeAntioxidative Power (AP)Power (AP)

• AP method: the reducing activity against the stable test radical DPPH (diphenyl-picryl-hydrazyl) is monitored by ESR (Electron Spin Ressonance) spectroscopy.

( )

AntioxidativeAntioxidative Power (AP)Power (AP) • The signal intesity decay is recorded at different times until saturation, when allthe Lipochroman-6 molecules have reacted with DPPH.

• AP parameter: allows to quantify both the reaction capacity and speed ofantioxidants. AP and tr (reaction time) are typical values for each antioxidant.

with an excellent long term stability

is a powerful antioxidantAfter being stored, Lipochroman-6 enhanced its AP up to 1,470,000, and reaction time values ranged from 0.20 (at 0h) to 0.17 (at 48h).

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (V)

4. Determination of the 4. Determination of the AntioxidativeAntioxidativePower (AP) Power (AP) vsvs BHT in final formulations (I)BHT in final formulations (I)

• AP method: the reducing activity against the stabletest radical DPPH (diphenyl-picryl-hydrazyl) ismonitored by ESR (Electron Spin Ressonance)

( )

Power (AP) Power (AP) vsvs BHT in final formulations (I)BHT in final formulations (I) monitored by ESR (Electron Spin Ressonance)spectroscopy.

• The signal intesity decay is recorded at differenttimes until saturation, when all the Lipochroman-6molecules have reacted with DPPH.

• AP parameter: allows to quantify both the reactioncapacity and speed of antioxidants. AP and tr (reactiontime) are typical values for each antioxidant.

Sample AP (AU) tr (min)

LIPOCHROMAN-6 995,424 0.19,

BHT 12,157 4.52

showed an antioxidative power 82-fold higher than BHTshowed an antioxidative power 82 fold higher than BHT

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (VI)

4. Determination of the 4. Determination of the AntioxidativeAntioxidativePower (AP) Power (AP) vsvs BHT in final formulations (II)BHT in final formulations (II)

• Long-term stability of two antioxidants(Lipochroman-6 or BHT) incorporated in finalcosmetic creams at 0 05% was determined by

( )

Power (AP) Power (AP) vsvs BHT in final formulations (II)BHT in final formulations (II) cosmetic creams at 0.05% was determined bymeasuring AP and tr.

• Measurements were performed over 3 monthsat different storage conditions (RT and 40 ºC).

showed a 35-47-folds higher AP comparedto the cream containing BHT, at RT

dand

29-34-folds higher than BHT, at 40 ºC

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (VII)

5. Singlet oxygen quenching ability5. Singlet oxygen quenching ability • Among ROS, there is an exceptionally reactive form known assinglet oxygen (02(a1∆g)).

( )

• The near-infrared emission of 02(a1∆g) was detected at1275nm by a NIR photomultiplier module working in photoncounting mode.

• Test solutions were prepared by mixing 2.5ml of methanol-d4,100µl of stock 1H phenalen 1 one solution (photosensitiser) and

Singlet oxygen decay vs Lipochroman-6 concentration100µl of stock 1H-phenalen-1-one solution (photosensitiser) anda known volume of a Lipochroman-6 solution in methanol-d4.Lifetime of O2(a1∆g) decay is affected by Lipochroman-6

i t t i gl t g gis a potent singlet oxygen scavenger

Its activity is remarkably close to tocopherols, which rank among the most effective molecules.

IN VITRO EFFICACY –MOLECULAR-LEVEL STUDIES (VIII)

6. Inhibition of peroxide formation in essential oils6. Inhibition of peroxide formation in essential oils

( )

• Peroxidation is a very frequent chain reaction = lipid molecules + oxygen (ROS).

• Essential oils are susceptible of autoxidation when in contact with oxygen due to their high lipid content.

• Essential oils were dissolved in isohexadecane and 0.01% Lipochroman-6 was added.

• Samples were kept at 60 ºC for 2 weeks.

Rosemary (RosmarinusOfficinalis) Leaf Oil

Oleic Acid)

Control

0.01%

completely inhibited peroxide formation in both oils

IN VITRO EFFICACY –CELLULAR-LEVEL STUDIES (I)( )

1. Cellular 1. Cellular photoprotectionphotoprotection(Comet Assay)(Comet Assay)

• Melanocytes were incubated with three differentconcentrations of Lipochroman-6 (1.0µg/ml, 10.0 µg/ml(Comet Assay)(Comet Assay) and 50.0 µg/ml) for 2 hours at 37 ºC. After this contactperiod, cells were irradiated with UVA.

• UV-induced DNA breaks were analyzed by the alkalineComet Assay.

• Negative controls included non-irradiated untreated cellsand non-irradiated cells treated with Lipochroman-6. UVAirradiated cells without Lipochroman-6 were used aspositive controls.UVA treated cell

(positive control)Non treated cell

(negative control)

The broken DNA migrates from the cellNo DNA migration

Cells + UVA+ 1.0 µg/mL LIPOCHROMAN-6

Cells + UVA+ 10.0 µg/mLLIPOCHROMAN-6

Cells + UVA+ 50.0 µg/mL LIPOCHROMAN-6

Small DNA migration

19.3% PROTECTIONSmall DNA migration

57.7% PROTECTION

Small DNA migration

72.2% PROTECTION

prevents skin from photoaging

Protects cellular DNA from ROS oxidation, induced by UVA radiation.

IN VITRO EFFICACY –CELLULAR-LEVEL STUDIES (II)

2. Inhibition of oxidative stress on human dermal fibroblasts2. Inhibition of oxidative stress on human dermal fibroblasts

( )

• Oxidative stress is the imbalance between cellularproduction of free radical species and the ability of cells toeliminate them employing endogenous antioxidant defencemechanisms. This stress damage cells irreversibly.

I ki ll lt id ti t t d b th• In skin cell cultures, oxidative stress was generated by theaddition of H2O2 to the culture medium.

• The protecting effects of the tested compounds(Lipochroman-6, Resveratrol, Vitamin E and Ferulic Acid)were measured by a cell viability assay (Calcein AM assay)were measured by a cell viability assay (Calcein-AM assay).

is more effective than Resveratrol,Vitamin E and Ferulic Acid against oxidative stress

COSMETIC BENEFITS

COMPLETE PROTECTION from Photoagingfrom Photoaging

A ti ROS & Cell viability increase against oxidative stress

Cellular photoprotection

Anti-ROS & Anti-RNS

Ideal ingredient for skin-care formulations

More effective than Not only protects the

• Double activity

It has a very strong antioxidative

More effective than Resveratrol

Not only protects the skin, but also

formulations from oxidationy g

capacity according to AP method (Gematria Test Lab GmbH)

TECHNICAL INFORMATION

DESCRIPTIONChromane that protects cells from several damages such as structural Chromane that protects cells from several damages such as structural alteration of proteins, inhibition of enzymatic activity and interferences of the regulatory cellular function.

APPEARANCEAPPEARANCEPowder.

INCIINCIDimethylmethoxy chromanol.

PROPERTIESPROPERTIESProtects cells from reactive species, preventing skin from premature aging.

APPLICATIONSLipochroman-6 can be incorporated in lipophilic based cosmetic formulations to avoid deterioration of skin.

DOSAGEDOSAGE0.01-0.05%

AN ACTIVE SHELTER TO PROTECT CELLS

Disclaimer:

While the claims and supporting data provided in this publication are believed to be reliable and they arepresented free and for guidance only, there are no warranties of any kind. All expressed and impliedwarranties are disclaimed The recipient is solely responsible for ensuring that products marketed to

All tradenames, trademarks, copyrights and images used herein belong to their respective and lawful ownerswarranties are disclaimed. The recipient is solely responsible for ensuring that products marketed to

consumers comply with all relevant laws and regulations. LIPOTEC is the exclusive holder of the bothindustrial and intellectual property rights identified herein. Recipient of this publication agrees toindemnify and hold harmless each entity of the LIPOTEC organization for any and all regulatory actionarising from recipient’s use of any claims or information in this publication, including, but not limited to,use in advertising and finished product label claims, and not present this publication as evidence of finishedproduct claim substantiation to any regulatory authority.

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