V. 1

STEP BY STEP TO PERFECTLY EVEN

SKIN TONE

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BRIGHT AND EVEN SKIN TONE NEED

• Aesthetic models of most cultures involve presenting an even skin tone.

• In addition, Asian ideal skin implicates a light complexion, as a pale skin is more desirable.

Improvement of the skin tone is a global demand whether for lightness or evenness

Hyperpigmented areas or dark spots appear as signs of aging, being the first ones in Asian skin, even before wrinkles.

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MELANIN IS KEY FOR SKIN PIGMENTATION

Changes in deposition of melanin lead to a loss of evenness of the skin complexion

Skin pigmentation

• E.g. solar lentigines (or age spots), due to UV damage.

Changes deposition with

time

Hyperpigmented areas that lead to reduced evenness.

Melanin

• Protects from ultraviolet (UV) radiation.

• Determines skin color, influencing tone and luminosity.

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PRODUCTION OF MELANIN IN THE SKIN

• Melanin synthesis and deposition is controlled the melanin epidermal unit:

30-40 keratinocytes

1 melanocyte

UV radiation stimulates keratinocytes

producing signaling factors that activate

melanocytes

melanogenesis and transfer of melanin to

keratinocytes

Pigmentation

MELANIN EPIDERMAL UNIT

+

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1. Activation of melanocytes. (UV radiation, inflammation, hormones, …)

2. Formation and maturation of melanosomes.

4. Transport and transfer to keratinocytes.

3. Synthesis of melanin in melanosomes.

SKIN PIGMENTATION INVOLVES MULTIPLE STEPS

• The process of melanin production and distribution in the epidermis involves several steps:

1

2 3

4

By acting on all steps, skin pigmentation can be extensively modulated

Melanocyte

1

2 3

4

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ACTIVATION OF MELANOCYTES

• Multiple extracellular molecules, secreted by several skin cells, bind to receptors in the melanocyte surface and induce signaling pathways leading to MITF* (key transcription factor) activation and expression of melanogenic genes.

MITF

Melanosome development

Endothelin-1 Wnt

Endothelin B receptor

Melanogenic genes Melanin synthesis

Frizzled receptor

Activated melanocytes lead to melanosomes formation and

melanin synthesis

* MITF (Microphthalmia-associated transcription factor)

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MELANOSOMES DEVELOPMENT

• Melanosomes, the cytoplasmic organelles where melanin biosynthesis takes place follow 4 stages of maturation:

MAT

URA

TIO

N

I

II

III

IV

• EARLY MELANOSOMES

Formation of a fibrillar matrix (Pmel17 and melan-A).

Incorporation of melanin-synthesizing enzymes.

• LATE MELANOSOMES

Melanin synthesis.

Melan-A and Pmel17 are structural proteins involved in formation and maturation of melanosomes

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MELANIN PRODUCTION IN MELANOSOMES

Melanin-synthesizing enzymes:

Tyrosinase (TYR): rate-limiting enzyme.

Tyrosinase-related proteins (TRP): TRP-1 and -2, collaborate with TYR in subsequent steps.

TYR, TRP-1 and TRP-2 are the enzymes responsible for brown-black pigment synthesis

Two types of melanin are produced:

Eumelanin: black-brown pigment.

Pheomelanin: yellow-reddish pigment.

• Melanin biosynthesis pathway takes place inside melanosomes.

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TRANSPORT AND TRANSFER OF MELANIN

• Mature melanosomes with melanin are transported along melanocyte dendrites, in a movement mediated by cytoskeletal components.

Epidermal keratinocytes incorporate melanin by

phagocytosis of melanosomes

• Then, melanosomes are transferred to keratinocytes by phagocytosis, that can be inhibited by DKK1*.

* DKK1 (Dikkopf-related protein 1)

Keratinocyte

DKK1

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MELANIN, UV & DNA DAMAGE

• Melanin has a role in photoprotection due to its ability to absorb and scatter UV radiation.

Different types of DNA damage

(crosslinks, adducts, breaks, …)

Specific DNA repair

pathways

Accumulation of DNA damage alters cell functions, leading to changes in pigmentation and other photoaging signs

Keratinocyte DNA

Melanosomes shield the nucleus from UV radiation.

• Nevertheless, UV causes direct and indirect DNA damage:

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A DNA REPAIR SYSTEM

• Nucleotide excision repair (NER) is critical for the repair of UV-induced DNA damage,

with the following process:

DNA repair is essential to prevent the accumulation of damage and preserve skin properties

1. Recognition of DNA damage with RNA polymerase, ERCC6 and ERCC8*.

2. DNA unwinding by helicases.

3. Excision of damaged DNA through XPG‡ and XPF-ERCC1*.

4. New DNA synthesis mediated by PCNA¥ and RFCΔ.

* ERCC1, ERCC6 and ERCC8 (Excision-repair cross-complementing proteins). ‡ XPG (Xeroderma pigmentosum, complementation group G). # XPF (Xeroderma pigmentosum, complementation group F). ¥ PCNA (Proliferating cell nuclear antigen). ∆ RFC (Replication factor C).

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STEP BY STEP TO PERFECTLY EVEN SKIN TONE

Biotechnological extract that acts on various steps of the pigmentation process to brighten and homogenize skin complexion.

• Modulates gene expression of melanogenic proteins.

• Decreases levels of melan-A involved in melanosomes development.

• Reduces the amount and activity of tyrosinase.

• Diminishes the rate of melanin production by melanocytes.

• Inhibits phagocytosis, to limit melanin distribution in skin.

• Prevents hyperpigmentation induced by Wnt, related with age spots.

• Upregulates DNA repair genes.

• In vivo, brightens the skin and reduces contrast, size and melanin content of hyperpigmented areas.

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BRIGHLETTE™ marine ingredient EFFICACY

IN VITRO EFFICACY

• Modulation of the expression of melanogenic genes

• Decrease in melanosomes maturation

• Reduction in tyrosinase protein expression

• Inhibition human tyrosinase activity

• Melanin production decline in melanocytes

• Modulation of phagocytosis in keratinocytes

• Melanogenesis inhibition on reconstructed epidermis

• DNA repair pathway activation

IN VIVO EFFICACY

• Brightening and evening the skin complexion

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-49.3%

-9.4%

-24.0%

-37.4%

-32.1%

-35.1%

-34.1%

-53.8%

-60 -40 -20 0

TRP-2

TRP-1

TYR

Melan-A

Pmel17

FZD10

FZD8

EDNRB

Fold change (%)

GENE EXPRESSION OF MELANOGENIC COMPONENTS

• Human epidermal melanocytes were treated with 10 µg/mL BRIGHLETTE™ marine ingredient concentrate for 13 days. Then, the RNA was purified and analyzed using a microarray and fold variation in gene expression with respect to control was calculated.

Modulation of gene expression of melanogenic signaling pathways, melanosomal proteins and melanin-synthesizing enzymes.

BRIGHLETTE™ marine ingredient acts on multiple genes involved

in the pigmentation process

860.9%

0 200 400 600 800 1000

DKK1

Fold change (%)

Melanocyte activation

Melanosomes development

Melanin production

Melanosomes transfer

* EDNRD (Endothelin B receptor). ‡ FZD8 and FZD10 (Frizzled family receptor 8 and 10).

*

‡

‡

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-40.0%

****

*** **

0

0,5

1

1,5

2

Control Forskolin Kojic acid BRIGHLETTE™ marine ingredient concentrate

Fold

in

du

ctio

n o

f m

elan

-A p

rote

in

**p<0.01, ***p<0.001, ****p<0.0001

DECREASE IN MELANOSOMES MATURATION

• A co-culture of melanocytes and keratinocytes was established and incubated with 4 µg/mL forskolin (to induce melanosome maturation), 71 µg/mL kojic acid or 10 µg/mL active ingredient concentrate for 48 h.

• Then, melan-A protein was stained by immunofluorescence and its levels quantified. Non-treated cells were used as a control.

BRIGHLETTE™ marine ingredient targets melan-A to interfere with the development of melanosomes

Control Forskolin

Kojic acid BRIGHLETTE™ marine ingredient concentrate

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REDUCTION IN TYROSINASE PROTEIN EXPRESSION

• Human epidermal melanocytes were incubated for 13 days with 10 µg/mL BRIGHLETTE™ marine ingredient concentrate. Then, a cell-based enzyme-linked immunosorbent assay (ELISA) was performed to measure the quantity of tyrosinase protein in the cells.

Non-treated cells were used as a control.

Tyrosinase protein within melanocytes was reduced by up to 48.9% with BRIGHLETTE™ marine ingredient

****p<0.0001

****

****

0

20

40

60

80

100

120

Control BRIGHLETTE™ marine ingredient concentrate

(10 µg/mL)

BRIGHLETTE™ marine ingredient concentrate

(50 µg/mL)

Tyr

osi

nas

e p

rote

in le

vels

(%

)

-48.9% -29.4%

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44.7% ****

58.6% ****

24.3% *

36.3% **

0

10

20

30

40

50

60

70

Control Kojic acid(100 µg/mL)

Kojic acid(160 µg/mL)

BRIGHLETTE™ marine ingredient concentrate

(10 µg/mL)

BRIGHLETTE™ marine ingredient concentrate

(100 µg/mL)

Inh

ibit

ion

of

hu

man

tyr

osi

nas

e ac

tivi

ty (

%)

INHIBITION OF HUMAN TYROSINASE ACTIVITY

A reduction of human tyrosinase activity of 36.3% was observed.

*p<0.05, **p<0.01, ****p<0.0001

Anti-tyrosinase activity that minimizes the potential to produce pigment

• A human tyrosinase assay kit was used to measure the formation of a DOPAchrome complex.

• The reaction mix was incubated with different concentrations of kojic acid (positive control) or BRIGHLETTE™ marine ingredient concentrate and the tyrosinase enzyme.

• Absorbance was read at 490 nm using a microtiter plate reader.

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MELANIN PRODUCTION DECLINE IN MELANOCYTES

• Human epidermal melanocytes were treated with BRIGHLETTE™ marine ingredient concentrate, for 13 days.

• Melanin was extracted from the cells and absorbance read at 450 nm. Melanin concentration was determined from a standard curve and normalized by cell number.

Non-treated cells were used as a control.

Cells treated with BRIGHLETTE™ marine ingredient contained notably less melanin

**** ****

****

0

20

40

60

80

100

120

Control BRIGHLETTE™ marine ingredient concentrate

(1 µg/mL)

BRIGHLETTE™ marine ingredient concentrate

(5 µg/mL)

BRIGHLETTE™ marine ingredient concentrate

(10 µg/mL)

Mel

anin

co

nce

ntr

atio

n (

%)

-61.3% -54.3% -46.3%

****p<0.0001 Control

BRIGHLETTE™ marine ingredient concentrate (10 µg/mL)

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MODULATION OF PHAGOCYTOSIS IN KERATINOCYTES (I)

• Human epidermal keratinocytes (HEKa) were incubated for 30 min with:

100 ng/mL DKK1, as a positive control for phagocytosis inhibition. HEKa treated with medium alone were used as a control (DKK1-non treated).

supernatants from human epidermal melanocytes treated with 1 µg/mL BRIGHLETTE™ marine ingredient concentrate for 13 days. HEKa treated with supernatants from melanocytes incubated with medium alone were used as a control (supernatant of non-treated melanocytes).

DKK1 (positive control for

phagocytosis inhibition) Melanocyte supernatant

Melanocyte BRIGHLETTE™ marine ingredient concentrate

Phagocytosis assay

HEKa

Phagocytosis assay

HEKa

• A microsphere-based phagocytosis assay was performed: HEKa were incubated for 4 h with fluorescent microspheres and, through confocal microscopy, internalization of the spheres was visualized.

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MODULATION OF PHAGOCYTOSIS IN KERATINOCYTES (II)

Internalization of particles by keratinocytes decreased due to the treatment of melanocytes with the active ingredient.

**p<0.01, ***p<0.001

BRIGHLETTE™ marine ingredient has an inhibitory effect on phagocytosis

Control (DKK1-non treated)

Control (supernatant of non-treated melanocytes)

Positive control (DKK1-treated)

Supernatant of melanocytes treated with BRIGHLETTE™

marine ingredient concentrate

Green color: keratinocytes outlines; Red color: fluorescent microspheres.

***

-69.0%

**

0

0,2

0,4

0,6

0,8

1

1,2

Control(DKK1-non treated)

Positive control(DKK1-treated)

Control (supernatant ofnon-treated melanocytes)

Supernatant of melanocytes treated with BRIGHLETTE™

marine ingredient concentrate

Fold

in

du

ctio

n o

f p

hag

ocy

tosi

s

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MELANOGENESIS INHIBITION ON RECONSTRUCTED EPIDERMIS (I)

• Reconstructed human pigmented epidermis (phototype IV) was incubated for 5 days with medium alone (control), 200 ng/mL Wnt-1 (to induce hyperpigmentation) and Wnt-1 with BRIGHLETTE™ marine ingredient concentrate.

• Melanin was detected and quantified through a Fontana-Masson stain on tissue sections and optical microscopy.

Control Wnt-1

Wnt-1 + BRIGHLETTE™ marine ingredient concentrate (100 µg/mL)

Wnt-1 + BRIGHLETTE™ marine ingredient concentrate (200 µg/mL)

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MELANOGENESIS INHIBITION ON RECONSTRUCTED EPIDERMIS (II)

A reduction in melanin content of 52.1% was observed in epidermis models treated with Wnt-1.

BRIGHLETTE™ marine ingredient prevents hyperpigmentation

related to age spots

**

* *

0

0,5

1

1,5

2

2,5

Control Wnt-1 Wnt-1 + BRIGHLETTE™ marine ingredient

concentrate (100 µg/mL)

Wnt-1 + BRIGHLETTE™ marine ingredient

concentrate (200 µg/mL)

Fold

-in

du

ctio

n o

f m

elan

in c

on

ten

t

-41.5% -52.1%

*p<0.05, **p<0.01

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DNA REPAIR PATHWAY ACTIVATION

Upregulated genes of the NER pathway of DNA repair in the epidermis, necessary due to the loss of protective pigment.

Expression of DNA repair genes is induced by BRIGHLETTE™ marine ingredient

• Reconstituted epidermis (phototype IV) was treated with medium alone (control) or 10 µg/mL BRIGHLETTE™ marine ingredient concentrate for 48 h.

• Then, RNA was extracted and purified from the tissue and analyzed using a microarray. Fold variation in gene expression with respect to control was calculated.

11.5%

9.1%

9.0%

7.0%

11.4%

11.3%

0 5 10 15

RFC4

RFC3

PCNA

ERCC1

ERCC8

POLR2F

Fold change (%)

DNA damage recognition

Excision of damaged DNA

DNA synthesis

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BRIGHTENING AND EVENING THE SKIN COMPLEXION (I)

• INCREASE OF L* AND ITAº (I)

A spectrophotometer was used to assess luminance (L*) and the individual typological angle (ITAº). The higher these parameters, the lighter the skin. Contrast between hyperpigmented and non-hyperpigmented skin was calculated.

• 22 Asian female volunteers, between 38-53 years old and with hyperpigmented regions on the skin applied a cream containing 2% BRIGHLETTE™ marine ingredient to half face and a placebo cream to the other half twice a day for 8 weeks.

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BRIGHTENING AND EVENING THE SKIN COMPLEXION (II)

• INCREASE OF L* AND ITAº (II)

Cross-polarized light UV-light

0 weeks

8 weeks

Hyperpigmented area Overall skin pigmentation

Cross-polarized light

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BRIGHTENING AND EVENING THE SKIN COMPLEXION (III)

BRIGHLETTE™ marine ingredient brightens and evens the skin

• INCREASE OF L* AND ITAº (III)

L* and ITAº increased in normal and in hyperpigmented areas. ITAº contrast of dark spots decreased by 12.7%.

2.5% *** 2.1%

***

-3.9% -4

-2

0

2

4Hyperpigmented

Non-hyperpigmented Contrast

L* v

aria

tio

n (

%)

15.3% ***

7.0% ***

* -12.7%

-15

-10

-5

0

5

10

15

20Hyperpigmented

Non-hyperpigmented Contrast

ITA

º va

riat

ion

(%

)

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BRIGHTENING AND EVENING THE SKIN COMPLEXION (IV)

The area occupied by hyperpigmented regions decreased by 6.9%, an effect that was 19.5% better than the placebo treatment.

Dark spots are smaller and less visible after treatment with

BRIGHLETTE™ marine ingredient

• SIZE REDUCTION OF HYPERPIGMENTED SPOTS

Variation in the area of dark spots was assessed by image analysis of digital photographs using appropriate software.

0 weeks 8 weeks

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BRIGHTENING AND EVENING THE SKIN COMPLEXION (V)

The amount of pigment was diminished by 61.1%.

BRIGHLETTE™ marine ingredient reduces the melanin in hyperpigmented spots

• EFFECT ON MELANIN CONTENT IN DARK SPOTS

The amount of melanin on hyperpigmented regions was measured by reflectance confocal microscopy.

0 weeks 8 weeks

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CONCLUSIONS

biotechnological marine ingredient that acts on multiple stages of the pigmentation process.

modulated the expression of melanogenic genes and induces DNA repair related genes.

reduced melan-A protein levels (40.0%), thus interfering with development of melanosomes.

diminished the amount of human tyrosinase protein by up to 48.9% and its activity by 24.3%.

decreased the amount of melanin pigment produced by melanocytes in culture by 61.3%.

acting on melanocytes, resulted in a 69.0% lower uptake of particles (phagocytosis) in keratinocytes.

neutralized Wnt-induced hyperpigmentation (related to age spots) in reconstituted epidermis.

applied at 2% in vivo, provided brightness by increasing L* (2.5%) and ITA⁰ (15.3%), and decreased contrast of dark spots, leading to a great improvement in evenness.

shrank darks spots area (6.9%) and decreased their melanin content by 61.1%.

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DESCRIPTION Extract obtained by biotechnology from a marine microorganism isolated from the southwest coast of the Tenerife island (Spain). BRIGHLETTE™ marine ingredient acts on multiple stages of the pigmentation process to consistently reduce the deposition of melanin in the epidermis. As a consequence it brightens the skin complexion, especially hyperpigmented spots, greatly contributing to increase evenness APPEARANCE Transparent solution containing 0.11% Plankton Extract. INCI Butylene Glycol, Water (Aqua), Plankton Extract. Preservative free. PROPERTIES BRIGHLETTE™ marine ingredient acts on various stages of melanin production and deposition in the skin for an integral and effective control of pigmentation. It improves luminosity and decreases visibility of hyperpigmented areas to even out the skin tone. APPLICATIONS BRIGHLETTE ™ marine ingredient can be used in any cosmetic formulation to provide a brighter complexion and in anti-aging products to even the skin tone by reducing dark spots. DOSAGE 2%

pH Recommended pH range between 4.0 and 7.0.

TECHNICAL INFORMATION

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STEP BY STEP TO PERFECTLY EVEN SKIN TONE

BIOINTEC™ and BRIGHLETTE™ are owned by The Lubrizol Corporation.

The other tradenames and trademarks used herein belong to their respective and lawful owners.

Note: Graphs and photographs of efficacy tests are available for customer use provided that the final product contains the same concentration of active as the formulations in our tests. Customers must request written permission for use of the graphic material and/or ingredient tradenames to Lubrizol. Customers are responsible for compliance with local and international advertising regulations.

The specific situation of the trademark in each country may vary and we recommend that you contact us for updated information.

Disclaimer:

The information including the claims and supporting data provided in this publication are provided for informational purposes only, upon the express condition that the User make its own assessment of the appropriate use of such information. While the information contained herein is believed to be reliable, there are no representations, guarantees, or warranties of any kind made as to its accuracy, suitability for particular applications, how the product(s) will perform in combination with other substance or in the User’s process or the results obtained. All expressed and implied warranties are disclaimed. Lubrizol and its affiliates MAKE NO WARRANTIES, EXPRESS OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. The User is solely responsible for ensuring that products marketed to consumers comply with all relevant laws and regulations and assumes all risk and liability of any use or handling of any materials. User agrees to indemnify and hold harmless Lubrizol and its affiliates for any and all actions arising from User’s use of any information including claims in this publication, including, but not limited to, use in advertising and finished product label claims, and not present this publication as evidence of finished product claim substantiation to any regulatory authority. It is the User’s sole responsibility to determine if there are any issues relating to patent infringement relating to the supplied information. Nothing contained herein is to be considered as permission, recommendation, nor as an inducement to practice any patented invention without permission of the patent owner.