AN ACTIVE SHELTER TO
PROTECT CELLS
v5
WHERE IS THE MARKET GOING TO?
9 Niche segments, such as teenagers and men, are growing in the skin care
market.
9 They are also following the trends settled by women’s skin care.
9 PREVENTION is a rising worry.
Teenagers
…beginning
beginning preventive care are
in favor of prevention rather
than waiting until it’s too late!
Men
More advanced grooming products:
mass and premium brands are
offering 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
g on your
y
skin TODAY will minimise its damage
g TOMORROW
knows: that acting
WHERE IS THE MARKET GOING TO?
Big players are more concerned with
prevention and how important it is.
RADICAL SCAVENGING – CLAIM OR
REALITY?
IN THE SKIN
Healthy skin is rich in an intrinsic antioxidative defense system.
system
In old skin or under oxidative stress, this defense
system becomes more and more deficient.
deficient
IN THE MARKET
Skin care products with antioxidative claims are one of the most fast growing market
for cosmetics worldwide.
* Gematria Test Lab GmbH – AP
method (HPC Today, n 1/2009)
… BUT 73% off products
d t that
th t claim
l i
antioxidative
ti id ti
properties showed no or very low antioxidative activity! *
Adding SPF and UVA protection is a major trend in the skin care market, but not the
only one…
We are finding
g more and more p
products in the skin care market
with 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
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.
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…
requires NEW TERMINOLOGY:
ROS
Reactive Oxygen Species
RNS
R
Reactive
ti Nit
Nitrogen
g SSpecies
i
RCS
Reactive Carbonyl Species
REACTIVE SPECIES
External Factors
RCS
Lipid Peroxidation
Protein Crosslinking
ROS
DNA damage
RNS
Internal Factors
Cellular necrosis
ROS AND RNS
ROS (REACTIVE OXYGEN SPECIES) AND OXIDATIVE STRESS
• U
Under
d normall conditions,
di i
ROS are cleared
l
d ffrom the
h cell
ll b
by endogenous
d
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.
RNS (REACTIVE NITROGEN SPECIES)
• Nitrosative stress occurs when the generation of RNS in a system exceeds
the system
system’ss ability to neutralize and eliminate them.
• Nitrosative stress may lead to nitrosylation reactions (with NO•) that can
alter protein structure thus inhibiting normal function.
NITRIC OXIDE (NO•)
NITRITE (NO2-)
NITRIC DIOXIDE (NO2•)
PEROXYNITRITE (ONOO-)
NITROXYL ANION (NO-)
LIPOCHROMAN-6: VERSATILITY
Li
LipochromanLipochroman
h
-6,
6 due
d to iits two reactive
i centers, h
has a double
d bl action:
action
ti
ROS scavenger
RNS scavenger
8
CH3O
1
O
7
HO
2
3
6
5
4
Lipochroman-6
IN VITRO EFFICACY –
MOLECULAR-LEVEL STUDIES (I)
1. RNS Capture
• The nitration of tyrosine residues of proteins is an irreversible
reaction which compromises cyclic interconversion between
phosphorylated/non-phosphorylated
tyrosine,
necessary
in
activation/deactivation of enzymes and receptors.
• Evaluation of reactivity between tyrosine and peroxynitrite at
different concentrations of Lipochroman-6, determined by HPLC.
O
O
OH
peroxynitrite
O2N
OH
NH2
HO
NH2
HO
Tyrosine
3-Nitrotyrosine
p
p
Lipochroman-6
protects
enzymes from inactivation
Inhibits the reaction between tyrosine and
peroxynitrite in a dose
dose-dependent
dependent manner.
RNS
IN VITRO EFFICACY –
MOLECULAR-LEVEL STUDIES ((II))
2. ROS Capture (I)
• The ability of Lipochroman-6 to inhibit lipid peroxidation
was evaluated in incubations of sprague dowley rat
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
>1000
1000
Lipochroman-6 proved to inhibit lipid peroxidation at
lower
concentrations,
compared
l
i
d to other
h anti-ROS
i ROS
ROS
IN VITRO EFFICACY –
MOLECULAR-LEVEL STUDIES ((III))
2. ROS Capture (II)
• The antioxidant activity of Lipochroman-6 was evaluated
as a part of a study for an interlaboratory comparison to
assess the antioxidant potential of several compounds1.
ROS
TBA assay (2-Thiobarbituric acid)
Rank
1 J.
Substance
Mean
1
LIPOCHROMAN--6
LIPOCHROMAN
-0.38
0 38
2
BHT
-0.06
3
Trolox
0.23
3
Tocopherol
0.31
3
4-MBC
0.38
Protection of lipid structures
Buenger et al, International Journal of Cosmetic Science, 2006, 28, 135–146
was the best of five antioxidants
in the interlaboratory comparison.
IN VITRO EFFICACY –
MOLECULAR-LEVEL STUDIES ((IV))
3. Determination of the
Antioxidative Power (AP)
• AP method: the reducing activity against the stable test radical DPPH (diphenylpicryl-hydrazyl) is monitored by ESR (Electron Spin Ressonance) spectroscopy.
• The signal intesity decay is recorded at different times until saturation, when all
the Lipochroman-6 molecules have reacted with DPPH.
• AP parameter: allows to quantify both the reaction capacity and speed of
antioxidants. AP and tr (reaction time) are typical values for each antioxidant.
with an excellent long term stability
is a powerful antioxidant
After 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 Antioxidative
Power (AP) vs BHT in final formulations (I)
• AP method: the reducing activity against the stable
test radical DPPH (diphenyl-picryl-hydrazyl) is
monitored by ESR (Electron Spin Ressonance)
spectroscopy.
• The signal intesity decay is recorded at different
times until saturation, when all the Lipochroman-6
molecules have reacted with DPPH.
• AP parameter: allows to quantify both the reaction
capacity and speed of antioxidants. AP and tr (reaction
time) 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
82-fold
fold higher than BHT
IN VITRO EFFICACY –
MOLECULAR-LEVEL STUDIES ((VI))
4. Determination of the Antioxidative
Power (AP) vs BHT in final formulations (II)
• Long-term stability of two antioxidants
(Lipochroman-6 or BHT) incorporated in final
cosmetic creams at 0.05%
0 05% was determined by
measuring AP and tr.
• Measurements were performed over 3 months
at different storage conditions (RT and 40 ºC).
showed a 35-47-folds higher AP compared
to the cream containing BHT, at RT
and
d
29-34-folds higher than BHT, at 40 ºC
IN VITRO EFFICACY –
MOLECULAR-LEVEL STUDIES ((VII))
5. Singlet oxygen quenching ability
• Among ROS, there is an exceptionally reactive form known as
singlet oxygen (02(a1∆g)).
• The near-infrared emission of
02(a1∆g) was detected at
1275nm by a NIR photomultiplier module working in photon
counting mode.
Singlet oxygen decay vs Lipochroman-6 concentration
Lifetime of O2(a1∆g) decay is affected by Lipochroman-6
• Test solutions were prepared by mixing 2.5ml of methanol-d4,
100µl of stock 1H-phenalen-1-one
1H phenalen 1 one solution (photosensitiser) and
a known volume of a Lipochroman-6 solution in methanol-d4.
i a potent
is
t t singlet
i gl t oxygen
g scavenger
g
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 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 (Rosmarinus
Officinalis)) Leaf Oil
Oleic Acid
Control
0.01%
completely inhibited peroxide formation in both oils
IN VITRO EFFICACY –
CELLULAR-LEVEL STUDIES (I)
()
1. Cellular photoprotection
(Comet Assay)
• Melanocytes were incubated with three different
concentrations of Lipochroman-6 (1.0µg/ml, 10.0 µg/ml
and 50.0 µg/ml) for 2 hours at 37 ºC. After this contact
period, cells were irradiated with UVA.
• UV-induced DNA breaks were analyzed by the alkaline
Comet Assay.
Non treated cell
(negative control)
No DNA migration
UVA treated cell
(positive control)
• Negative controls included non-irradiated untreated cells
and non-irradiated cells treated with Lipochroman-6. UVA
irradiated cells without Lipochroman-6 were used as
positive controls.
The broken DNA migrates from the cell
Cells + UVA+ 1.0 µg/mL
LIPOCHROMAN-6
Cells + UVA+ 10.0 µg/mL
LIPOCHROMAN-6
Cells + UVA+ 50.0 µg/mL
LIPOCHROMAN-6
Small DNA migration
Small DNA migration
Small DNA migration
19.3% PROTECTION
57.7% PROTECTION
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 fibroblasts
• Oxidative stress is the imbalance between cellular
production of free radical species and the ability of cells to
eliminate them employing endogenous antioxidant defence
mechanisms. This stress damage cells irreversibly.
• In
I skin
ki cell
ll cultures,
lt
oxidative
id ti stress
t
was generated
t d by
b the
th
addition 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
(Calcein AM assay).
assay)
is more effective than Resveratrol,
Vitamin E and Ferulic Acid against oxidative stress
COSMETIC BENEFITS
COMPLETE PROTECTION
from Photoaging
Anti-ROS
A
ti ROS &
Anti-RNS
Cell viability
increase against
oxidative stress
More effective than
Resveratrol
• Double activity
It has a very
y strong
g antioxidative
capacity according to AP method
(Gematria Test Lab GmbH)
Cellular
photoprotection
Ideal ingredient
for skin-care
formulations
Not only protects the
skin, but also
formulations from
oxidation
TECHNICAL INFORMATION
DESCRIPTION
Chromane that protects cells from several damages such as structural
alteration of proteins, inhibition of enzymatic activity and interferences of
the regulatory cellular function.
APPEARANCE
Powder.
INCI
Dimethylmethoxy chromanol.
PROPERTIES
Protects cells from reactive species, preventing skin from premature aging.
APPLICATIONS
Lipochroman-6 can be incorporated in lipophilic based cosmetic formulations
to avoid deterioration of skin.
DOSAGE
0.01-0.05%
AN ACTIVE SHELTER TO PROTECT CELLS
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presented free and for guidance only, there are no warranties of any kind. All expressed and implied
warranties are disclaimed.
disclaimed The recipient is solely responsible for ensuring that products marketed to
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product claim substantiation to any regulatory authority.
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belong
g to their respective
p
and lawful owners