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

Welcome to Japan! Welcome to Tokyo! Recepción a Japón !! BUENOS AIRES 2010.9.21

The Novel Hair Dyeing Technology by Using Melanin Precursor Prepared by an Aspergillus Tyrosinase Koike, Kenzo 1 & Hata, Yoji 2 1 Kao Corporation, Tokyo, Japan. 2 Gekkeikan Sake Company, Kyoto, Japan. Traditional Japanese Sake Brewers

1. Background of Our Hair Research Hair Research encountered Biotechnology in 2001.

R e l a t i o n s h i p b e t w e e n m e l a n i n a n d c o l o r o f t h e h a i r - Melanin type and amounts affect color and brightness of the hair - Less Melanin Blond Bright Red Brown Dark More Black Melanin Eumelanin Rich Pheomelanin Rich Asian hair has more Eumelanin

R e l a t i o n s h i p b e t w e e n m e l a n i n a n d c o l o r o f t h e h a i r - Melanin type and amounts affect color and brightness of the hair - Less Melanin Blond Bright Red Brown Dark More Black Melanin Eumelanin Rich Pheomelanin Rich Asian hair has more Eumelanin

R e l a t i o n s h i p b e t w e e n m e l a n i n a n d c o l o r o f t h e h a i r - Melanin type and amounts affect color and brightness of the hair - Less Melanin Blond Bright Red Brown Dark More Black Melanin Eumelanin Rich Pheomelanin Rich Asian hair has more Eumelanin

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) Gray hair Black hair No melanin Melanin Granules

M e l a n o g e n e s i s E u m e l a n i n b i o 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. O H O tyrosinase O O 2 H O 2 C N H 2 O 2 O H O H tyrosine N H O H Eumelanin O H tyrosinase N H H O 2 C N H 2 5,6-Dihydroxyindole O 2 DOPA O CO 2 O O tyrosinase O dopaquinone H O 2 C N H O O H 2 dopa - dopaquinone O O H N H O 2 H O 2 C + N H N H H O 2 C H O C cyclodopa 2 O H dopachrome （DHICA) O H G. Prota ., “Progress in the N H chemistry of melanins and related H O 2 C 5,6-Dihydroxyindole metabolites.” Med. Res. Rev . 8, -2-carboxylic acid 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. O H O tyrosinase O O 2 H O 2 C N H 2 O 2 O H O H tyrosine N H O H Eumelanin O H Melanin precursors tyrosinase N H H O 2 C N H 2 5,6-Dihydroxyindole O 2 DOPA O CO 2 O O tyrosinase O dopaquinone H O 2 C N H O O H 2 dopa - dopaquinone O O H N H O 2 H O 2 C + N H N H H O 2 C H O C cyclodopa 2 O H dopachrome （DHICA) O H G. Prota ., “Progress in the N H chemistry of melanins and related H O 2 C 5,6-Dihydroxyindole metabolites.” Med. Res. Rev . 8, -2-carboxylic acid 525-556, 1988

A n e w i d e a f o 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 o r ) Melanin Melanin precursor O Into Hair (Cuticle) N H O O H O N H O N H O O 2 (Air) H O N O H 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

1 3 Japanese Sake manufacturing Japanese Sake manufacturing Japanese Traditional Biotechnology Rice 酵母（ 麹（ KOBO) KOJI) Saccharomyces Aspergillus oryzae cerevisiae (yeast) fungas) 日本酒 Japanese SAKE 酒造り (SAKE Dukuri) Alcohol Fermentation Clean Water Traditional Brand from 17th century (400 yrs)

Koji Black Koji (Normal) (Irregular ) There was a color problem, occasionally, during 1950s and1960s.

F u n g a l m e l a n i n f o r m a t i o n i n t h e p r o c e s s o f f e r m e n t a t i o n . ＜ SAKE Fermentation ＜ SAKE Fermentation ＞ ＞ Rice Steamed rice seed seed KOJI Water Yeast MOROMI strained Koji Black Koji sake (Normal) (Irregular ) sludge SAKE 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 oryzae and Its Over expression in Solid-State Culture (Rice Koji)” J. Biosci. Bioeng . 97 400-405 2004.

T y r o s i n a s e w a s f o u n d t o b e e x p r e s s e d i n B l a c k K o j i . Black Koji Tyrosinase (enzyme) The Japanese Sake company found a tyrosinase strongly expressed in Black Koji. Koji : Fermented rice with Aspergillus oryzae Solid Liquid New Collaboration research Surface Culture was started Culture 1.Obata et al , “Cloning of a Novel Tyrosinase-Encoding Gene (melB) from Aspergillus oryzae and Its Over expression in Solid-State Culture (Rice Koji)” J. Biosci. Bioeng . 97 400-405 2004.

M e l a n o g e n e s i s E u m e l a n i n b i o 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. O H O Which should be produced? tyrosinase O O 2 H O 2 C N H 2 O 2 O H O H tyrosine N H O H Eumelanin O H Melanin precursors tyrosinase N H H O 2 C N H 2 5,6-Dihydroxyindole O 2 DOPA O CO 2 O Plant- O tyrosinase originated O dopaquinone H O 2 C N H O O H 2 dopa - dopaquinone O O H DOPA N H O 2 H O 2 C + N H N H H O 2 C H O C cyclodopa 2 O H dopachrome （DHICA) O H N H H O 2 C 5,6-Dihydroxyindole -2-carboxylic acid

T h e d y e i n g a b i l i t y o f m e l a n i n p r e c u r s o r s 3 0 2 5 ） ΔE 2 0 染色性（ 1 5 Dyeing ability 1 0 5 0 D H I C A D H I o l e C A D H I o l e 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 o t a l s y s t e m f o r m e l a n i n p r e c u r s o r p r o d u c t i o n • Tyrosinase is produced by Production of catalyst fermentation. (tyrosinase) wash catalyst solution culture • Dopa is converted to Production of D O P A Dihydroxyindole in tyrosinase melanin precursor process. solid-liquid solution of separation melanin precursor enzyme reaction • The product is adjusted for Adjustment of solution cosmetics materials. concentration and adjustment 5,6-Dihydroxyindole solution

M e l a n i n p r e c u r s o r p r o d u c t i o n b y f u n g u s T y r o s i n a s e Melanin precursor was produced from Plant-originated Dopa. Melanin Precursor production DOPA (Plant-origin) D O P A M e l a n i n Melanin precursor (DHI) p r e c u r s o r H O H O N H • 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 H 2 O 2 – Gentle to hair: less damage – Easy to use: no mixing, no long waiting.

F o r m u l a t i o n o f t h e H a i r D y e Foamer Dihydroxyindole (surfactant) (New Dyestuff) Alkali Solvents Thickeners Antioxidants Foam type: Ease of Use Aerosol: maintain stability by Oxygen shutoff 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 o n s h i p b e t w e e n D H I c o n c . a n d D y e i n g a b i l i t y 6 0 5 0 ) △E ( 4 0 y t i l i Dyeing image b 3 0 a g n i 2 0 D H I 0 . 3 % e y D H I 0 . 1 % D 1 0 0 0 1 2 3 4 5 6 DHI: 0.1% or 0.3%＋3％ ammonia C y c l e n u m b e r 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.