NEUROLIGHT.61 GNeuro-active solution to treat dark spots

BE INSPIRED…

Pancratium maritimum

Neurolight.61 G: extracted from Pancratium maritimum

• Synonyms: Star Lily, Lys de Mer (French) or Sea Daffodil

• One of the most beautiful of the wild Cretan flowers

• Found on most of the northern beaches of Chania.

• Pancratium: comes from the Greek and literally means “All Strength”, perhaps referring to the supposed tonic properties of the plant or to the strength a plant must have to tolerate the extreme climate of the hot and dry summer beach.

Maritimum: comes from Latin and means “from the sea-coast”

View of Chania Island

Pancratium maritimum

Pancratium maritimum: description

Monocotyledonous herbaceous perennial that flowers during the hottest months of summer.

Flowers:

– pure white and very large up to 15 cm long

– have a sweet exotic lily scent. It is said that the strong perfume of the flowers keep sheep away from the coasts

– open at nightfall and close the following afternoon

Pancratium maritimum: habitat

• Maritime sands on the sandy beaches in the immediate vicinity of the sea

• Direct exposure to the sun and the sea winds and to continuous sprays of salt water droplets carried by the wind

• Famous in Crete, we also find it on Mediterranean coasts and on the islands of Hoedic and Houat in Brittany

Hoedic Island

Houat Island

Pancratium maritimum: legend

• This elegant white flower was the inspiration behind Minoan artists who depicted its beauty in the Palace of Knossos on Crete. Knossos is the largest Bronze Age archaeological site on Crete and probably the ceremonial and political centre of the Minoan civilization and culture.

• In Knossos Fresco Sea-daffodils are associated with the veneration of a goddess.

• Pancratium maritimum was also a plant associated with the vegetation goddess Persephone (or Ariadne), who each year had to pass through all the phases of life, including the underworld.

• In Christian times, daffodils became a symbol of resurrection

Minoan Sea Daffodils “Lilies” Fresco

Representation of Persephone

The production of Neurolight.61 G

• Pancratium maritimum is a protected specie, its harvest is therefore totally forbidden

• Codif R&N has developed Pancratium cultures in greenhouse

• Neurolight.61 G is obtained by aqueous extraction followed by purification step

• Neurolight.61 G duly complies to our Controlled Management program of Natural Resources

OBSERVE…

Dark spots, origin and formation

Dark spots: what they are, where they come from

• Dark spots are a group of keratinocytes, full of melanin secreted by up-regulated melanocytes

• Dark spots are characteristics of ageing skins and photo-damaged epidermis which have both accumulate a lot of internal stress

• Recent studies have demonstrated the implication of two intrinsic factors in dark spots formation:

1. Molecules of internal stress 2. Neuropeptides (small proteins released by nerves)

(1) Toyoda and al,. Epidermal innervation correlates with severity of photodamage. A quantitative ultrastructural study. Exp Dermatol. 1996 Oct; 5(5):260-6

(2) Masahiko and al,. Calcitonin Gene-Related Peptide Upregulates Melanogenesis and Enhances Melanocyte Dendricity via Induction ofKeratinocyte-Derived Melanotrophic Factors. Journal of Investigative Dermatology Symposium Proceedings 4:116–125, 1999

1. Dark spots & Internal StressDescription of the pathway

1. External stress, by generating free radicals, create stressful conditionsfor keratinocytes

2. The stress protein: p53 is secreted to activate the protection mechanisms of the cell

3. Among other molecule, p53 activate the expression of POMC (Pro-Opio MelanoCortine)

4. The activation of POMC is directly followed by the release of a-MSH and ACTH which is then partially transformed in a-MSH

5. a-MSH binds its receptors at the surface of the melanocytes and trigger melanin synthesis

6. Melanin is then transported to the keratinocytes to protect the skin from UVs

1. Dark spots & Internal StressHow to counter-balance stress effect

NO: inhibiting p53.

p53 is the sentinel of the cell, it activates repairing and protection mechanisms. We therefore cannot act on this protein

YES: Inhibiting POMC expression

And check the inhibition of global activation of melanogenesis

1. Dark spots & internal stress:Neurolight.61 G inhibits POMC and melanin synthesis

-65%-70

-60

-50

-40

-30

-20

-10

0POMC

Exp

ress

ion

% c

om

par

ed to

the

con

tro

l

0.33% Neurolight.61 G inhibits POMC

0.33% Neurolight.61 G inhibits melanogenesis

Neurolight.61 G inhibits the activation of melanogenesis related to internal stress

Gene expression: analyzed by RT-qPCR on B16 melanocytes cultivated without (control) or with 0.33% of Neurolight.61 G. Melanin synthesis: analyzed on B16 melanocytes stimulated by a-MSH 60nM and cultivated without (control) or with Neurolight.61 G.

Control Neurolight.61 G

2. Dark spots & NeuropeptidesDescription of melanocyte environment

Thanks to their dendrites, melanocytes communicate with:

• keratinocytes: melanin is exported to keratinocytes thanks to the dendrites

• nerves: they release messengers called neuropeptides which bind receptors at the surface of the dendrites to activate melanin synthesis and/or exportation

2. Dark spots & NeuropeptidesLink between nerves and dark spots

• In 1996, Toyoda and al put in evidence a correlation between degree of epidermal innervations and chronic photo-damages.

• In photo-damages skins, communication between nerves and melanocytes are increased

• Among others, the neuropeptide Substance P could activate melanine release when binding its receptor TacR1 at the surface of melanocyte’s dendrites

Codif’s laboratories have demonstrated the implication of Substance P in melanin release by melanocytes

• By decreasing the length of dendrites

• By decreasing the synthesis of Substance P receptors

Both actions will decrease the communication between nerves and melanocytes and thus melanin release

2. Dark spots & Neuropeptideshow to counter-balance the effect of neuropeptides

(1) Toyoda and al,. Epidermal innervation correlates with severity of photodamage. A quantitative ultrastructural study. Exp Dermatol. 1996 Oct; 5(5):260-6

(2) Masahiko and al,. Calcitonin Gene-Related Peptide Upregulates Melanogenesis and Enhances Melanocyte Dendricity via Induction ofKeratinocyte-Derived Melanotrophic Factors. Journal of Investigative Dermatology Symposium Proceedings 4:116–125, 1999

3B

2. Dark spots & Neuropeptides Neurolight.61 G decreases the length of dendrites

Dendricity: observed on human melanocytes cultivated with 200µM IBMX; without or with Neurolight.61 G.

Without Neurolight.61 G With 0.033% Neurolight.61 G

0.033% Neurolight.61 G decreases the dendricity of melanocytes by 10%

2. Dark spots & Neuropeptides Neurolight.61 G inhibits the synthesis of TacR1

Inhibition of TacR1 (Substance P Receptor)

-50%

-60

-50

-40

-30

-20

-10

0

Expr

essi

on %

com

pare

d w

ith th

e co

ntro

l

Gene expression: analyzed by RT-qPCR on B16 melanocytes cultivated without (control) or with 0.33% of Neurolight.61 G.

In addition to inhibit the dendricity of melanocytes, Neurolight.61 G also inhibits the synthesis of

Substance P receptor at the surface of dendrites

2. Dark spots & Neuropeptides Neurolight.61 G inhibits the stimulation of melanin release by

Substance P

-38%**

+24%*

0

2

4

6

8

10

12

14

Control 1nM Substance P 1nM Substance P + 0,16%Neurolight.61 G

Mel

anin

Con

cent

ratio

n (µ

g/w

ell)

0.16% Neurolight.61 G significantly inhibits the release of melanin triggered by Substance P, by -38%

*p<0.05 Student test

**p<0.01 Student test

Culture of B16 melanocytes cultivated with 30µM IBMX

Conclusion on in-vitro tests

2 origins for dark spots formation:

• Cellular origin: photo-damaged skins = stressed keratinocytes = activation of melanin synthesis via POMC and a-MSH• Neural origin: photo-damaged skins = high innervations degree = activation of the release of melanin by neural factors like Substance P

By inhibiting both pathways, Neurolight.61 G inhibits melanin synthesis (intra-cellular melanin) and melanin release (extra-cellular melanin)

-62%*

-73%*

0102030405060708090

100

Intra-cellular melanin Extra-cellular melanin

Mel

anin

Con

cent

ratio

n (µ

g/m

l)

Control0,33% Neurolight.61 G

*p<0.05 Student test

Melanogenesis is stimulated by addition in the culture medium of 60nM alpha-MSH

-62%

-47%

-35%

-70

-60

-50

-40

-30

-20

-10

0b-Arbutin Kojic acid Neurolight.61 G

% o

f inh

ibiti

on

Comparison of Neurolight.61G with reference molecules on extracellular melanin

Each molecule has been tested on B16 melanocytes cultures

b-Arbutin 0.003%

% of b-Arbutin usually used in in-vitro test: 0.001 to 0.005%

Kojic Acid 0.005%% of kojic acid usually used in in-vitro test: 0.001 to 0.005%

Neurolight.61G 0.33%Equivalent to 0.001% of dry matter

REVEAL…

Clinical tests

PROTOCOLE

• 84 days; realized in Thailand on 16 volunteers, phototype III-IV

• Twice daily application of a cosmetic cream containing 1.5% of Neurolight.61 G

• Measured parameter: digital cross-polarized photograph of one randomly selected hand; chromametric method and ITA evaluation; and self-assessment questionnaire

• Skin pigmentation is evaluated using a chromameter that measures two parameters:

– the Luminance L*: measurement of the clarity of the skin, from dark to pale.

– the Chrominance b*: defines the range of blues to yellows.

These parameters are studied to measure the Individual Topographical Angle (ITA), which defines the degree of pigmentation of the skin of an individual.

1.5% Neurolight.61 G erases dark spots

1.5% Neurolight.61 G significantly decreases the pigmentation of dark spots

1.5% Neurolight.61 G lights the pigmentation of dark spots without lightening the pigmentation of the skin

Lightening effect on pigmentary spots after 84 days

+12,1%*

up to +61,5%

0

10

20

30

40

50

60

70

Mean value Max Value

ITA

Var

iatio

n (%

of D

0)

1.5% Neurolight.61 G significantly lights the pigmentation of dark spots by 12% on average and up to 61% without

lightening the skin

*p<0.05 Student test

Decrease of the difference of pigmentation spots/skin after 84 days

-11,6%

up to -53%

-60

-50

-40

-30

-20

-10

0Mean value Max Value

ITA

Varia

tion

(% o

f D0)

D0 D42 D84

1.5% Neurolight.61 G significantly reduces the size of dark spots

• by -18.9% and up to -63.4% after 42 days

• by -24.8% and up to -61.1% after 84 days

1.5% Neurolight.61 G reduces the surface of dark spots

1.5% Neurolight.61 G reduces the surface of dark spots

*p<0.05 Student test

1.5% Neurolight.61 G reduces the size of dark spots

• by -18.9% and up to -63.4% after 42 days

• by -24.8% (significantly) and up to -61.1% after 84 days

-24,8%*

-18,9%

up to -61,1%up to -63,4%

-70

-60

-50

-40

-30

-20

-10

0D42 D84

Varia

tion

in %

Mean valueMax value

Conclusion on Neurolight.61 G

Neurolight.61 G is a neuro-active ingredient that significantly reduce the area and the pigmentation of dark spots thanks to an original & innovative action mechanism:

- inhibition of melanin synthesis triggered by stressed keratinocytes

- inhibition of melanin release triggered by neural factor: Substance P

This ingredient put in evidence, for the first time, the role of substance P in the release of melanin by dendritic melanocytes

Recommended use: 1.5%Ecocert and Cosmos approved & One of the few flower extract QDA registered

EU/US Inci name: Glycerin (and) Water (and) Pancratium maritimum extract

Chinese Inci name: Glycerin (and) Water (and) Glucose (and) Mineral salts (and) Mannose (and) Bioflavonoids