Recepcin a Japn !! BUENOS AIRES 2010.9.21 The Novel Hair Dyeing - - PowerPoint PPT Presentation

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BUENOS AIRES 2010.9.21

The Novel Hair Dyeing Technology by Using Melanin Precursor Prepared by an Aspergillus Tyrosinase

Traditional Japanese Sake Brewers

Koike, Kenzo 1 & Hata, Yoji 2 1 Kao Corporation, Tokyo, Japan. 2 Gekkeikan Sake Company, Kyoto, Japan.

• 1. Background of

Our Hair Research

Hair Research encountered Biotechnology in 2001.

R e l a t i

• n

s h i p b e t w e e n m e l a n i n a n d c

• l
• r
• f

t h e h a i r

• Melanin type and amounts affect color and brightness of the hair -

Brown Blond Black Red

Less Melanin Bright Dark More Melanin

Eumelanin Rich Pheomelanin Rich

Asian hair has more Eumelanin

R e l a t i

• n

s h i p b e t w e e n m e l a n i n a n d c

• l
• r
• f

t h e h a i r

• Melanin type and amounts affect color and brightness of the hair -

Brown Blond Black Red

Less Melanin Bright Dark More Melanin

Eumelanin Rich Pheomelanin Rich

Asian hair has more Eumelanin

R e l a t i

• n

s h i p b e t w e e n m e l a n i n a n d c

• l
• r
• f

t h e h a i r

• Melanin type and amounts affect color and brightness of the hair -

Eumelanin Rich

Brown Blond Black Red

Pheomelanin Rich

Asian hair has more Eumelanin

Less Melanin Bright Dark More Melanin

T h e d i f f e r e n c e b e t w e e n G r a y a n d B l a c k H a i r

• The difference is only melanin -

Male hair sample from a Japanese (50’s)

No melanin Melanin Granules

Gray hair Black hair

M e l a n

• g

e n e s i s E

u m e l a n i n b i

• s

y n t h e s i s

• Eumelanin is a polymer made from Tyrosine or DOPA.
• This pathway is initiated by Tyrosinase oxidation reaction.
• Melanin precursors are converted to Melanin by Oxygen.

N H

2

H O

2C

O H O H N H

2

H O

2C

O H N H H O

2

C O H O H O O N H

2

H O

2C

N H O O H O

2C

N H O H O H N H O O N H O O H O

2C

N H O H O H H O

2C

+

• tyrosinase

O2

Eumelanin

tyrosine

DOPA dopaquinone cyclodopa dopachrome 5,6-Dihydroxyindole 5,6-Dihydroxyindole

• 2-carboxylic acid

dopaquinone dopa

（DHICA) tyrosinase tyrosinase

O2 O2 O2 CO2

• G. Prota., “Progress in the

chemistry of melanins and related metabolites.” Med. Res. Rev. 8, 525-556, 1988

Melanogenesis

Eumelanin biosynthesis

• Eumelanin is a polymer made from Tyrosine or DOPA.
• This pathway is initiated by Tyrosinase oxidation reaction.
• Melanin precursors are converted to Melanin by Oxygen.

N H

2

H O

2C

O H O H N H

2

H O

2C

O H N H H O

2

C O H O H O O N H

2

H O

2C

N H O O H O

2C

N H O H O H N H O O N H O O H O

2C

N H O H O H H O

2C

+

• tyrosinase

O2

Eumelanin

tyrosine

DOPA dopaquinone cyclodopa dopachrome 5,6-Dihydroxyindole 5,6-Dihydroxyindole

• 2-carboxylic acid

dopaquinone dopa

（DHICA) tyrosinase tyrosinase

O2 O2 O2 CO2

Melanin precursors

• G. Prota., “Progress in the

chemistry of melanins and related metabolites.” Med. Res. Rev. 8, 525-556, 1988

A n e w i d e a f

• r

h a i r d y e i n g s y s t e m w i t h m e l a n i n ( p r e c u r s

• r

)

Melanin precursor Melanin

N H O H O H

N H N H N H O O O O O O

Into Hair (Cuticle) O2 (Air)

Benefits

・ Melanin and its precursors are biological substances. ・ The Melanin precursor is small enough to penetrate hair. ・ It converts to melanin by exposure to air. ・ Melanin has a natural dark color.

Issues： The Melanin precursor is very unstable. None of the natural compounds were industrially available.

By the way, we have traditional biotechnology in Japan.

Fungus and yeast have been used in food manufacturing and various fermentation processes in Japan for over 1000 years.

Hair Research encountered Biotechnology in 2001.

• 02. Preparation of Melanin

Precursor using fungus Tyrosinase

Manufacturing new ingredient from natural resources using same process as in nature.

Collaboration between Kao & Gekkeikan

Japanese Sake manufacturing Japanese Sake manufacturing

1 3

Japanese Traditional Biotechnology

Rice

Saccharomyces cerevisiae (yeast) Aspergillus oryzae

fungas)

酒造り (SAKE Dukuri) Alcohol Fermentation

酵母（ KOBO) 麹（ KOJI)

Clean Water

日本酒 Japanese SAKE Traditional Brand from 17th century (400 yrs)

Black Koji (Irregular) Koji (Normal)

There was a color problem,

• ccasionally, during 1950s and1960s.

F u n g a l m e l a n i n f

• r

m a t i

• n

i n t h e p r

• c

e s s

• f

f e r m e n t a t i

• n

.

Black Koji (Irregular) Koji (Normal)

strained sake sludge

＜ ＜SAKE Fermentation SAKE Fermentation＞ ＞

Rice

Steamed rice

KOJI

Yeast seed MOROMI SAKE seed

Water

Melanin formation

As a result, the Black Koji was identified, and the Melanin formation mechanism was found.

Tyrosinase from Aspergillus oryzae makes melanin.

1.Obata et al, “Cloning of a Novel Tyrosinase-Encoding Gene (melB) from Aspergillus

• ryzae and Its Over expression in Solid-State Culture (Rice Koji)” J. Biosci. Bioeng. 97

400-405 2004.

T y r

• s

i n a s e w a s f

• u

n d t

• b

e e x p r e s s e d i n B l a c k K

• j

i .

Tyrosinase (enzyme)

The Japanese Sake company found a tyrosinase strongly expressed in Black Koji. Black Koji Koji : Fermented rice with Aspergillus oryzae Solid Surface Culture Liquid Culture

New Collaboration research was started

1.Obata et al, “Cloning of a Novel Tyrosinase-Encoding Gene (melB) from Aspergillus

• ryzae and Its Over expression in Solid-State Culture (Rice Koji)” J. Biosci. Bioeng. 97

400-405 2004.

M e l a n

• g

e n e s i s E

u m e l a n i n b i

• s

y n t h e s i s

• Eumelanin is a polymer made from Tyrosine or DOPA.
• This pathway is initiated by Tyrosinase oxidation reaction.
• Melanin precursors are converted to Melanin by Oxygen.

N H

2

H O

2C

O H O H N H

2

H O

2C

O H N H H O

2

C O H O H O O N H

2

H O

2C

N H O O H O

2C

N H O H O H N H O O N H O O H O

2C

N H O H O H H O

2C

+

• tyrosinase

O2

Eumelanin

tyrosine

DOPA dopaquinone cyclodopa dopachrome 5,6-Dihydroxyindole 5,6-Dihydroxyindole

• 2-carboxylic acid

dopaquinone dopa

（DHICA) tyrosinase tyrosinase

O2 O2 O2 CO2

Melanin precursors

Plant-

• riginated

DOPA Which should be produced?

T h e d y e i n g a b i l i t y

• f

m e l a n i n p r e c u r s

• r

s

5 1 1 5 2 2 5 3

D H I C A D H I

• l

e C A D H I

• l

e

染色性（ ΔE ）

Dyeing ability

DHI

DHICA：5,6-Dihydroxyindline 2-carboxylic acid DHIoleCA ：5,6-Dihydroxyindole 2-carboxylic acid DHI ：5,6-Dihydroxyindole (DHI) Dyeing condition： 3%Ammonia, 0.1% each compound, 3 times

T h e t

• t

a l s y s t e m f

• r

m e l a n i n p r e c u r s

• r

p r

• d

u c t i

• n
• The product is adjusted for

cosmetics materials.

• Tyrosinase is produced by

fermentation.

• Dopa is converted to

Dihydroxyindole in tyrosinase process.

culture wash catalyst solution solution of melanin precursor solid-liquid separation enzyme reaction concentration and adjustment 5,6-Dihydroxyindole solution

Production of catalyst (tyrosinase) D O P A Production of melanin precursor Adjustment of solution

M e l a n i n p r e c u r s

• r

p r

• d

u c t i

• n

b y f u n g u s T y r

• s

i n a s e

DOPA (Plant-origin) Melanin precursor (DHI)

N H O H O H

Melanin Precursor production

M e l a n i n D O P A p r e c u r s

• r

Melanin precursor was produced from Plant-originated Dopa.

• DOPA (the starting material) is of Plant-origin.
• Melanin precursor was produced by using the same process as in nature.
• The Melanin precursor is a sustainable compound.

• 03. Application of Melanin

Precursor to Hair Dye

Development of a natural coloring product for gray hair.

New Concept of Natural Hair Dye

• The new dyestuff, 5,6-dihydroxyindole,has

been manufactured from natural resources using a similar process to nature.

• It ensures “True Natural Hair Color” by

recovering color by melanin.

• Air oxidation system without H2O2

– Gentle to hair: less damage – Easy to use: no mixing, no long waiting.

F

• r

m u l a t i

• n
• f

t h e H a i r D y e Foam type: Ease of Use Aerosol: maintain stability by Oxygen shutoff Foamer (surfactant) Dihydroxyindole (New Dyestuff) Alkali Solvents Thickeners Antioxidants

How to Use: 20g of the product was applied to hair. After standing for 5 min. hair is washed with shampoo.

T h e r e l a t i

• n

s h i p b e t w e e n D H I c

• n

c . a n d D y e i n g a b i l i t y

1 2 3 4 5 6 1 2 3 4 5 6 C y c l e n u m b e r D y e i n g a b i l i t y ( △E ) D H I . 3 % D H I . 1 %

Dyeing image

DHI: 0.1% or 0.3%＋3％ ammonia Procedure: Chinese grey hair, RT, 5min, shampooed and dried, This treatment was repeated as indicated.

DHI at 0.1 - 0.3% dyed gray hair gradually.

T h e e f f e c t

• f

a l k a l i n e a n d p H i n D y e i n g a b i l i t y pH 10 with ethanolamine was most effective

アルカ リ と p H の影響 ３ 回染色

5 1 1 5 2 2 5 3 3 5 4 4 5 5 9 9 . 5 1 1 . 5 1 1

ｐ H

染色性⊿E

モノ エタ ノ ールアミ ン 4 8 % 苛性ソ ーダ 炭酸ナト リ ウム 炭酸グアニジン 2 8 % アンモニア

Dyeing ability

Ethanolamine 48% sodium hydroxide sodium carbonate guanidine carbonate 28%ammonia

Melanin precursor in the formulation was stable for 6 months

2 4 6 8 1 1 2 2 4 6 8 1 保存日数( 日) 染料（ ％） 5 ℃ R T 4 ℃

storage period (days)

DHI

B r

• w

n a n d A s h s h a d e s w e r e a c h i e v e d . Brown shade Ash shade

DHI: 0.3%＋1％ Ethanolamine + Antioxidants Procedure: Chinese grey hair, RT, 5min, shampooed and dried, This treatment was repeated up to 5 times.

M e l a n i n p r e c u r s

• r

i s c

• n

v e r t e d t

• M

e l a n i n b y a i r Dyeing ability: ±

5 min Just after discharging

Melanin formation by air

Dyeing ability:＋＋

Easy to use: no mixing, no long waiting period

T h e m e c h a n i s m f

• r

d y e i n g w i t h m e l a n i n p r e c u r s

• r

Cross section

• 1. Melanin precursor can penetrate into the cuticle of hair.
• 2. Melanin is formed by exposure to air.
• 3. Gray hair is gradually dyed by step-wise use (3-5 times).
• 4. The final color of hair then looks more natural.

Cortex

Cuticle Gray hair 3 times 5 times A part

Dyeing image How to Use

・ Treatment ・ 5 min. ・ Shampoo

D y e i n g a b i l i t y

Panelist results

Before 1st 3rd 5th

・ Each repeated treatment brought natural change. ・ The color of hair became very natural. ・ The color only faded after application was stopped.

A d v a n t a g e s

• f

t h e n e w t e c h n

• l
• g

y Hair damage level Skin staining

Cysteic acid [mol％]

. . 5 1 . 1 . 5 2 . 未処理 ﾒ ﾗ ﾆ ﾝ 前駆体 5 回 ﾍ ｱ ｶ ﾗ ｰ 5 回 ﾍ ｱ ﾏ ﾆ ｷ ｭ ｱ 5 回

Non Treated Direct Dye Hair Dye New Technology

New Technology Direct dye system

Skin → 30 min. → Washing with water Hair → Hydrolysis → A.A. analysis (HPLC)

Conclusions

A New Natural Hair Dye

• The new dyestuff from natural resources

– DHI has been manufactured using a similar process to nature.

• Restoring Melanin into gray hair.

– Recovery of natural color by melanin

• Gentle to hair and Easy to use

– Air oxidation system without H2O2

Gracias mucho!!

Thank you for your attention!

Gracias mucho!! Argentina!