Chia Wen ChenChia Wen Chen

Director of Natural ProductsDirector of Natural Products

Estee Lauder CompaniesEstee Lauder Companies

September 24, 2009September 24, 2009

SCC Education DaySCC Education Day

““Development of Development of

NaturalNatural Actives for Actives for

Spa and Cosmetic Spa and Cosmetic

ProductsProducts””

R&D Capabilities

Product development assistance (scale up from a bench top process)

Phytochemical analysis (to determine marker compound)

Full analytical support (method development)

Requirements

ISO 9001:2000 certified (cGMP)

Heavy Metal and Pesticide analysis

Safety test for topical application

Sustainable Biomass (wild craft, cultivation)

Certified Organic (NOP, EcoCert, IFOAM)

Lot to lot consistency: What are we really getting?

Microbial issues 10,000 cfu for dietary supplement vs 100 cfufor cosmetic

Heavy metal and Pesticide analysis – due to the pollution in soil and water

Regulatory (REACH) and Safety (Topical)

Patents- not patent protected

Converting an ingestible for topical use (need to define biological activity)

Challenges

Cosmetic Products

Are designed not only to moisturize the skin

and reduce the visibility of lines and

wrinkles…

But also to protect the skin from the

damages caused by environmental exposure.

TCTC

EC

FB

TC

MN

KC

UV

ILIL--11ααααααααPLAPLA22

TNFTNF

MMPMMP--11

PLAPLA2 2

Nitric oxide (NO)

Blood flow

Pain Irritant Allergen

NO

NO

AA PGE2

PL AA

PLA2

COX2

ILIL--11αα

PMN

IL-12

Collagenases

PG’sCAMs

UVB, ozone, smoke

PL

A2arachadonic acid

PGE2

cox2

neutrophil

vasodilation

macrophage

collagenaseelastase

UVBO3

O3

O3

Ozone

cigarette smoke

Dermiscapillary

collagen

Basement membraneEpidermis

O3

PGE2PGE2

neutrophil

Control of Inflammatory Reactions

Must take place at the very beginning of the cascade of reactions.

Inhibition of Phospholipase A2 will result in the control of the release of Arachidonic Acid, a major inflammatory mediator.

Additional anti-inflammatories will subsequently modulate the other steps of the cascade.

Insult (UV exposure, oxidative stress)

Activation of Phospholipase A2

Release of Arachidonic acid

Cyclo-oxygenase

Release of

Prostaglandins

Lipoxygenase

Release of LeukotrienesCytokines

(ILI,TNF ∝)

Recruitment of Leukocyte cells

Transport of Leukocytes by blood

Transfer to site of injury (chemotaxis)

Second oxidative burst and Protease (collagenase, elastase, tryptase)

Premature Aging

Poria Cocos

CentauriumResveratrol, hops

Rosemary, Citri reticulatae peel Grape seed extract

The Trend Toward Natural

Your consumers are watching and learning!

Consumer awareness is at an all time high.

Consumers want healthy products - and the industry is responding.

Natural ingredients and healthy lifestyle are attractive to consumers.

Morus spp.

Over 80% of the world's population uses plants as their primary medicines

500,000 plant

species

10,000substances per species

What makes plants so efficacious?

multi-taskers

� inpired by many years of folklore

� validated by science

� biodegradable and sustainable

;

High performance ingredientsFor high performance skin care

Finding new ingredients

Partnership with consultants and suppliers all over the world

Strategic alliance with academic research

OH

O

HO

OH O

OHO

HOOH

O

O

OH

O

HO OH OH

HO

rosavin salidroside

Rhodiola rosea

Treatment of skin cells with Rhodiola extract enhanced the ability of

the cells to survive after exposure to an external stress

(UV exposure)

0

20

40

60

80

100

30 60 90 120 150

UVB dose (mJ/cm2)

% s

urv

ival

untreated control

Rhodiola rosea

0

5

10

15

20

25

30

35

50 100 150 200

UVB dose (mJ/cm2)

nu

mb

er

of

da

ma

ge

d c

ell

s

untreated control

Rhodiola rosea

After 24 hours the number of damaged cells was

more then 2X lower in the Rhodiola treated skin model

Rhodiola rosea Enhances Rebound from Stress

ATP (energy depletion)

Mitochondrial DNA damageDNA damage

Cellular Damage (UVB, oxidative damage, inflammatory cytokines)

(-)Physiological effects

cancer

Animation 2

Animation 4

vs.

TREATED with Rhodiola rosea

Dark brown staining identifies heavily damaged skin cells. The

removal of damage was much faster in Rhodiola treated tissue.

Skin model stressed by simulated UVNo treatment

Rhodiola rosea Enhances Rebound from Stress

Mimosa tenuiflora

Mimosa bark extract with Flavonoids, tannins and saponins.

Anti-collagenase: EC50 = 80 ug/ml

Collagen synthesis: 31% at 0.0625 µg/ml and by 67% at 0.25 µg/ml,

Anti-collagenase Activity of Tepescohuite

EC50 = 80ug/ml

0.00

20.00

40.00

60.00

80.00

100.00

0 1.3 4 11.5 34 100 310 920

[tepescohuite] µg/ml

% i

nh

ibit

ion

Collagen Assay: Tepescohuite

0

2

4

6

8

10

12

Media 0.015625 0.0625 0.25

Dose (µg/ml)

Co

lla

ge

n (

µg

/ml)

Stratum

corneum

Langerhans

cell

melanocyteMast

cell

elastosis

collagen

elastin

Young skin Old skinPhotodamaged skin

Loss of collagen & elastin

Degraded collagen, thickened elastin

A B

Fragmented Fibrils

vs. Young cells Old cells

0

5000

10000

15000

20000

24 Yr 45 Yr 68 Yr

Cell

s/c

m2

In vitro cell proliferation as a function of age

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

24 Yr 45 Yr

Co

lla

ge

n P

rod

uc

ed

pe

r C

ell (

ng

)In vitro collagen production as a function of age

What makes a wrinkle

Stress induces damages to skin cells result in further loss of skin integrity – Wrinkle formation

Constant folding & unfolding of skin creates a weak point: the micro-tear.

Physical pressure on cells activates inflammatory reactions lead to premature degradation of collagen by collagenase

Wrinkle animation (4) skin folding Wrinkle animation (4) skin folding

development of inflammatory development of inflammatory

reactionreaction

Anti-Age: Dermatological Approach

Skin’s repair process activated by damaged skin cells. BENEFIT: improves skin

Inflammatory reaction degrades collagen & elastin. SIDE EFFECT: ages skin

Peels and lasers trigger cell’s natural defense mechanism.

Prevent : control collagenase, elastase and GAGasedegrading enzyme to prevent wrinkle formation.

Restore: collagen synthesis to accelerate “Healing” of skin

Repair:

help skin cells rebound from stress and prevent premature aging

Anti-Age: A Natural Approach

Healthy skin Aging/stressed skin

Collagen

Collagenase

Endogenous collagenase inhibitors

Collagen Fragmentation by Collagenase

ANIMATION 5b ANIMATION 5b collagenasecollagenase inhibitorinhibitor

ANIMATION 3 treatment of whole skin; fragments go to ANIMATION 3 treatment of whole skin; fragments go to

fibroblasts, activate collagen production. fibroblasts, activate collagen production.

Anti-Age: Wrinkle

Prevent premature destruction of key dermal components

Treatment:Anti-collagenase (Sigesbeckia, Tepescohuite)Anti-elastase (Boswellia, White Birch)Anti-GAGase (Silymarin, Centaurium)Collagen synthesis (Tepescohuite)

Result:Treatment of skin, most damaged by constant folding / unfolding.

Matricaria recutita flower

German Chamomile

Blue chamomile oil with confirmed:

5-LO inhibitory activity: EC50 = 50 ug/ml

Anti-chemotaxis: 97% inh. At 0.05%

LPO: EC50 = 0.003%

5-Lipoxygenase assaySCOPE

To analyse potential inhibitors of potato 5-lipoxygenase (5-LO).PRINCIPLE

5-lipoxygenase converts the substrate linoleic acid (diene 1-4) to a conjugated dieneproduct (diene 1-3), which can be detected at 234nm. A decrease in the absorbancesignal is indicative for an increased anti-5LO activity.

MATERIALNormal lab equipment

96 well half area plates with UV transparent bottom (Costar, 3679)8 channel multi pipette (20-200µl)

SpectrophotometerREAGENTS

H2O distilledPotassium dihydrogen phosphate (Acros, #205925000)

Dimethylsulfoxide (Acros, #12779)Potato 5-lipoxygenase (Cayman, #60400)

Linoleic acid 99% (Acros, #215040050)Nordihydroguaiaretic acid from Larrea divaricata (creosote bush) (approx. 95%)(reference inhibitor - NDGA) (Sigma, N1144)

Tween-20 (Janssen Chimica, #2333656)

5-Lipoxygenase Inhibition

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 10 20 30 40 50 60 70 80 90

[Blue chamomile oil organic (Hashem Brothers, #4905)] (µg/ml)

% Initial 5LO activity

IC50 ~50µg/ml

Chemotaxis Assay: Blue Chamomile Oil Organic

(BRD 4905)

0

20

40

60

80

100

120

Ctrl Caff .5% #4905 .005% #4905 .05%

PM

Ns

/HP

F

97% inhibition at 0.05%

LPO (Anti-oxidant)

0.000

0.001

0.001

0.002

0.002

0.003

0.003

30 min 120min

Conclusions

The development of new ingredients from plant

extracts involves many steps which can ultimately provide great benefits in cosmetics.

Despite the cost involved in such development, we believe that active ingredients from natural

sources provides the cosmetic industry with a quasi infinite source of ingredients which we are aiming at developing in the future.

Conclusion

Opportunity not a Headache

Carefully choose your partner

Know what you are working with

(Standardize extract)