““DevelopmentDevelopment ofof NaturalNatural ActivesActives forfor SpaSpa andand CosmeticCosmetic ProductsProducts ””

Chia Wen Chen Director of Natural Products Estee Lauder Companies September 24, 2009 SCC Education Day

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) Challenges

Lot to lot consistency: What are we really getting? Microbial issues 10,000 cfu for dietary supplement vs 100 cfu for 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) 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. Allergen UV Pain Irritant

COX2 IL -1α AA PGE2 KC

MN TNF PLA 2 ILIL --1αααα Collagenases MMP -1 PLA 2 (NO) IL-12 TC

PLA 2 PL AA NO PMN FB NO

TC TC CAMs Blood flow PG’s

EC Ozone UVB , ozone , smoke

PL A UVB arachadonic2 acid O O 3 3 cox 2 O cigarette smoke PGE 2 3 O3

Epidermis Basement membrane

PGE 2 PGE 2

neutrophil collagen

capillary Dermis collagenase elastase neutrophil

vasodilation 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) Poria Cocos Activation of Phospholipase A2 Resveratrol, hops Release of Arachidonic acid Centaurium Cyclo-oxygenase Lipoxygenase Release of Release of Leukotrienes Prostaglandins (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) Rosemary, Citri reticulatae peel Grape seed extract Premature Aging 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 as their primary medicines 500,000 10,000 substances species per species What makes plants so efficacious ?

multi-taskers

 inpired by many ; years of folklore

 validated by science

 biodegradable and sustainable High performance ingredients For high performance skin care Finding new ingredients

Partnership with consultants and suppliers all over the world Strategic alliance with academic research Rhodiola rosea

OH

HO O O O HO OH O

HO OH OH HO O HO O OH OH rosavin salidroside Treatment of skin cells with Rhodiola extract enhanced the ability of the cells to survive after exposure to an external stress (UV exposure)

100

80

60

untreated control 40 % survival Rhodiola rosea 20

0 30 60 90 120 150 UVB dose (mJ/cm 2) Rhodiola rosea Enhances Rebound from Stress

35 30 25 20 15 untreated control Rhodiola rosea 10 5

number of damaged cells 0 50 100 150 200 UVB dose (mJ/cm 2)

After 24 hours the number of damaged cells was more then 2X lower in the Rhodiola treated skin model Cellular Damage (UVB, oxidative damage, inflammatory cytokines)

ATP (energy depletion) DNA damage Mitochondrial DNA damage

cancer (-)Physiological effects Animation 2 Animation 4 Rhodiola rosea Enhances Rebound from Stress

Skin model stressed by simulated UV TREATED with No treatment Rhodiola rosea

vs.

Dark brown staining identifies heavily damaged skin cells. The removal of damage was much faster in Rhodiola treated tissue . 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 100.00 80.00 60.00 40.00 20.00 % inhibition 0.00 0 1.3 4 11.5 34 100 310 920

[tepescohuite] µg/ml Collagen Assay: Tepescohuite 12

10

8

6

Collagen (µg/ml) Collagen 4

2

0 Media 0.015625 0.0625 0.25 Dose (µg/ml)

Langerhans Stratum cell corneum elastosis

Mast melanocyte cell

elastin

collagen

Photodamaged skin Young skin Old skin Degraded collagen, thickened elastin Loss of collagen & elastin Young cellsvs. Old cells

A B

Fragmented Fibrils In vitro cell proliferation as a function of age

20000

15000 2

10000 Cells/cm

5000

0 24 Yr 45 Yr 68 Yr In vitro collagen production as a function of age

0.14

0.12

0.1

0.08

0.06

0.04

Collagen ProducedCollagen (ng) per Cell 0.02

0 24 Yr 45 Yr What makes a wrinkle

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 Stress induces damages to skin cells result in further loss of skin integrity – Wrinkle formation WrinkleWrinkle animationanimation (4)(4) skinskin foldingfolding developmentdevelopment ofof inflammatoryinflammatory reactionreaction Anti-Age: Dermatological Approach

Peels and lasers trigger cell’s natural defense mechanism.

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

Inflammatory reaction degrades collagen & elastin. SIDE EFFECT: ages skin Anti-Age: A Natural Approach

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

Restore : collagen synthesis to accelerate “Healing” of skin

Repair : help skin cells rebound from stress and prevent premature aging Collagen Fragmentation by Collagenase

Healthy skin Aging/stressed skin

Collagen

Collagenase

Endogenous collagenase inhibitors ANIMATIONANIMATION 5b5b collagenasecollagenase inhibitorinhibitor ANIMATION 3 treatment of whole skin; fragments go to fibroblasts, activate collagen production. Anti-Age: Wrinkle

Prevent premature destruction of key dermal components

Treatment: Anti-collagenase (, 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

Blue chamomile oil with confirmed: 5-LO inhibitory activity: EC50 = 50 ug/ml Anti-chemotaxis: 97% inh. At 0.05% LPO: EC50 = 0.003%

German Chamomile 5-Lipoxygenase assay

SCOPE To analyse potential inhibitors of potato 5-lipoxygenase (5-LO). PRINCIPLE 5-lipoxygenase converts the substrate linoleic acid (diene 1-4) to a conjugated diene product (diene 1-3), which can be detected at 234nm. A decrease in the absorbance signal is indicative for an increased anti-5LO activity. MATERIAL Normal lab equipment 96 well half area plates with UV transparent bottom (Costar, 3679) 8 channel multi pipette (20-200µl) Spectrophotometer REAGENTS H2O distilled Potassium dihydrogen phosphate (Acros, #205925000) Tween-20 (Janssen Chimica, #2333656) 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) 5-Lipoxygenase Inhibition

IC 50 ~50µg/ml 120.00

100.00

80.00

60.00

40.00 % Initial 5LO activity

20.00

0.00 0 10 20 30 40 50 60 70 80 90

[Blue chamomile oil organic (Hashem Brothers, #4905)] (µg/ml) Chemotaxis Assay: Blue Chamomile Oil Organic (BRD 4905)

120

100

80

60 PMNs/HPF 40 97% inhibition at 0.05% 20

0 Ctrl Caff .5% #4905 .005% #4905 .05% LPO (Anti-oxidant)

0.003 0.003 0.002 0.002 0.001 0.001 0.000 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)