p phyto tell pec calus tomestica (new version)

© Mibelle Biochemistry, Switzerland 2009

PhytoCellTecTM Malus DomesticaPlant stem cells for skin stem cell protection


PhytoCellTecTM Malus Domestica3 Stories

1. PCT Technologie

2. Swiss Apple Story

3. Stem Cell Cosmetics


PhytoCellTecTM Malus DomesticaInnovation Price

• BSB Innovation Price formost innovative natural active

• Received duringincosmetics 2008

• Patent pending in CH, EU, USA and Korea


1. Plant Cell Cultures

• New cell culture technology makes it now possible to cultivatecells of endangered and rare species

• Selected plant material (leave, fruit) is wounded to induce theformation of a callus tissue

• The callus of a plant consists of dedifferentiated, totipotentcells (plant stem cells)


The 3 Steps of PhytoCellTec

1. Preparing a sterile plant preparation2. Wounding the plant to induce callus tissue3. Growing callus cells in liquid cultures


Plant Cellculture Technology (PhytoCellTecTM =PCT)

• Suspension cultures from plant stem cells are grown in bioreactors

• Medium consists of sucrose, trace elements and plant hormones (cytokinins and auxins)

• Cultures grow with oxygen and without light (chlorophyll free)• Batch is harvested after all sugar has been metabolized• The cells are washed and homogenized to release the secondary

metabolites• Oil- and water-soluble ingredients are extracted by liposomal

structures


2. Swiss Apple Story


Storage Ability of Apples and Skin Aging

• Apples with very good storageabilities stay fresh over months

• This was an important factor forcultivar selection

• Modern apple cultivars are selectedfor intensive cultivation and for a sweet flavour since storage is no longer a problem

• Therefore cultivars with excellentstorage abilities disappeared fromthe market


Uttwiler Spätlauber

• An old cultivar is still alive in Switzerland but no longercultivated

• The Spätlauber variety derivesfrom the middle of the 18th century

• It was famous for its tremendous storability without shrinking

• Today this is a very rare and endangered apple variety withonly a few trees left in the world


Uttwiler Spätlauber

The importance of the UttwilerSpätlauber apple was honoredby the release of a stamp.


Young and Old


The Secret of Storage Ability

• High acid and tannin concentrations• Longevity of cells• Undiscovered factors?

This apple can only be used by PCT


PhytoCellTecTM Malus Domestica

CompositionMalus Domestica Stem Cells 9.0 %Phospholipids 0.14%Glycerin 0.4 %Phenoxyethanol 1.4 %Keltrol T 1.0 %Deionized water ad.100 %

CTFA/INCIMalus Domestica Fruit Cell Culture Extract (and) Xanthan Gum (and) Glycerin (and) Lecithin (and) Phenoxyethanol (and) Aqua/ Water


3. Stem Cell Cosmetics


What are Skin Stem Cells?

Properties of skin stem cells:

• Self-renewal - the ability to gothrough numerous cycles and celldivisions while maintaining theundifferentiated state

• Differentiation - the capacity to differentiate into other cell types of the same tissue

• Primary roles - replenish dying cellsand regenerate damaged tissues


Adult Stem Cells in Skin

In skin, different types of stem cells have been found:

Type Place

Epithelial skin stem cells Basal layer of the epidermis

Hair bulge stem cells Hair follicle


Epithelial Stem Cells

Stem cells are located in the basal layer where 2-7% of the cells are stem cells

Differentiating progeny of one epidermal stem cell

Epidermalstem cell

Transientamplifyingcells

BasementmembraneDermis

Basal layer

Spinous layer

Granular layer

Stratum corneum


Hair follicle matrix

Epidermal layer

Sebaceous gland

Hair shaft

Inner root sheath

Outer root sheath

Basal layer

Dermal papilla

Hair bulgestem cells

Infundibulum

Lineages derived from bulge stem cells

Differentiation pathways supplied by bulge stem cells

Hair Bulge Stem Cells


Stem Cells and Aging

• Stem cells have a limited life expection

• Environmental and intrinsicstress factors effect stem cells

• In aged skin the number and activity of stem cells is reduced

• Skin stem cells have to beprotected and supported to delay preliminary aging


Epigenetic FactorsComposition of Plant Stem Cells

• Plant stem cells contain similar epigenetic factors as human stem cells

• Plant stem cell extracts provide important nutrients to human stem cells

Plant stem cell nutrients improve the viability and healthiness of human stem cells


PhytoCellTecTM Malus DomesticaProvides two Different Modes of Activity

1. Use of plant stem cells to protect skin stem cellsThe components of plant stem cells such as Malus domestica are rich in specific factors relevant to protect and maintain the function of skin stem cells

epigenetic factors

2. Use of longevity cell extract to delay skin agingThe apple Uttwiler Spätlauber is known to contain ingredients which insure longevity of cells

metabolites


Protection of Stem Cells againstUV Damage – Study Design

Cell typeUmbilical cord blood stem cells

Study designIncubation for 24h with PhytoCellTec Malus Domestica Extract, UV radiation of cell cultures

Determination of living cellsMTS assay 48h after UV radiation

Control

+ 0.1% PhytoCellTecTM Extract


Protection of Stem Cells againstUV Damage – Results

S-142

PhytoCellTec Malus Domestica improves thevitality of stem cells

Increased protectionagainst UV stress


Novel Progenitor Cell Targeting Technology

• Cells: human epidermal stem cells (primary human keratinocytes) from a biopsie ca. 6% of cells are stem cells (optically not different from normal basal cells)

• Medium: specifically designed, fully defined medium that mimics the physiological environment of the in vivo stem cell niche

enrichment of so called keratinocyte progenitor cells (stem cells)

3x


Activity Assay for Epidermal Stem Cells

Stimulation of growth or over-expression of markers are not good parameters for the activity of stem cells

• Colony forming efficiency (CFE)Cell cultures are seeded at low density. The number of colonies formed is a value for the concentration and activity of progenitor/stem cells

+ apple stem cell extract

Incubation

Determination of CFE


0

20

40

60

80

100

0.01% 0.04%

Concentration of Malus Domestica stem cell extract

Incr

ease

of t

he C

FE c

om

par

ed to

co

ntro

l in

%

+ 92%

ResultNumber of active and healthy stem cells is increased compared to untreated culturesS-234

Increase of CFE byPhytoCellTecTM Malus Domestica


Result

PhytoCellTec apple stem cell extract enhanced CFE up to 92% compared to the control.

Maintenance of the stem cell vitality of epidermal stem cells was improved.


Growth of Hair Follicle

Hair follicles From human skin fragments (from micro dissection)

Test substance PhytoCellTecTM Malus Domestica 0.2%

Design Length of hair follicle increases until day 14 → after day 14 induction of senescence→ length of hair follicle decreases

Day 0 Day 4 Day 7

S-157


Length of Hair Follicle

-70

-60

-50

-40

-30

-20

-10

0

10

20

Day 16 Day 18 Day 20

Cha

nge

in le

ngth

co

mp

ared

to d

ay 1

4 in

%

Control 0.2% Malus Domestica stem cell extract

Senescence of hair (stem cells) can be delayed

S-157


PhytoCellTecTM Malus DomesticaEffect on Gene Expression in SenescentDermal Fibroblasts

Cell line Normal Human Dermal Fibroblasts

Design • H2O2 induced senescence (2h)followed by a treatment with2% PhytoCellTecTM Malus Domestica or not (144 h)

Parameters • Gene profile (microarray)


PhytoCellTecTM Malus DomesticaGene Profile (Microarray)

↑

↓ Gen, that is down-regulated by senescence induction (see control)

Gen, that is up-regulated or normalized in senescent cells by PhytoCellTec Malus Domestica

Gen After H2O2Control

After H2O2+ 2% Malus Domestica

stem cell extract

cyclin B1: induces proliferation 73 ↓ 130 ↑cyclin E1: cell cycle regulator 78 ↓ 135 ↑p53: tumor suppressor gene 63 ↓ 137 ↑insulin-like growth factor II: cell proliferation enhancer 71 ↓ 117 ↑

heme oxigenase 1: antioxidans enzyme 89 ↓ 211 ↑ S-194


In-Vivo Study: Anti-Wrinkle Effect of PhytoCellTecTM Malus Domestica

• Analysis of wrinkle depth in the crow‘s feet area• 20 volunteers• 28 days• Application twice daily• Against inital conditions



0

4

8

12

16

Day 14 Day 28

Dec

reas

e o

f wrin

kle

dep

th in

%

2% PhytoCellTec Malus Domestica

S-186



Day 0Day 282% PCT Malus Domestica

S-186


PhytoCellTecTM Malus DomesticaApplications

• Stem cell cosmetics• Rejuvenation products for face and

body• Fresh cell treatments• Anti-Aging formulations• Rejuvenation of hair follicles• Delay of hair aging


PhytoCellTecTM Malus DomesticaClaims

• Delays senescence of essential cells

• Protects longevity of skin stem cells• Protection of skin stem cells• Rejuvenation of skin• Maintains the power of regeneration• Decrease in wrinkles• Combats chronological aging• With plant stem cells to maintain

the power of regeneration• With plant stem cells to delay

senescence of essential skin cells


PhytoCellTecTM Malus DomesticaPlant stem cells for skin stem cell protection

Patent pending

p phyto tell pec calus tomestica (new version)

Documents

aging stem cells

adult stem cells

reduced skin stem cells

properties of skin stem

different types of stem

cells of endangered

pct mibelle biochemistry

old mibelle biochemistry