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Screening test for Development of Cosmeceuticals

Screening test for Development of Cosmeceuticals

Definition

Hybrids between cosmetics + pharmaceuticals A category of products placed between drugs and cosmetic

M. Amer, M. Maged, 2009

สารส�าคั�ญในตำ�าร�บเวชส�าอาง

Cosmeceutical Products

Cosmeceutical development

Define potentialsDefine potentialsDefine potentialsDefine potentials

OxygenOxygen

sensitivesensitiveSunSun

Burn Burn inflammatory inflammatory

Infection Infection

Hyperpigmentation Hyperpigmentation

Collagen and elastin degradation Collagen and elastin degradation

Skin dehydration Skin dehydration

Antioxidant assay

Cellular Oxidative stress

Cellular Oxidative stress

Antioxidant capacity

oxidative enzymeo Superoxideo Glutathione peroxidaseo Catalase

Antioxidant assay

Quantitative determination

Vitamin C Phenolics Carotenoids Glutathione

Antioxidant activity determination

DPPH Method FRAP Method ABTS Method ORAC Method

ว�ดคัวามสามารถในการก�าจั�ดอน�ม�ลอ�สระ โดยการถ�ายโอน Hอะตำอม จัากสาร antioxidant ไปย�งอน�ม�ลอ�สระ

Antioxidant activity determination

K. Thaipong et al. / Journal of Food Composition and Analysis 19 (2006) 669–675

Antioxidant activity determination

ABTS+ radical cation(colourless)

ABTS+ radical cation(colourless)

antioxidants

ABTS(Blue-green colour)

ABTS(Blue-green colour)

K. Thaipong et al. / Journal of Food Composition and Analysis 19 (2006) 669–675

Fe3+-TPTZ (uncolored) + reducing

antioxidant

Fe2 +-TPTZ (intense blue color )

K. Thaipong et al. / Journal of Food Composition and Analysis 19 (2006) 669–675

Antioxidant activity determination

K. Thaipong et al. / Journal of Food Composition and Analysis 19 (2006) 669–675

ว�ดคัวามสามารถในการเป!น reducing agent โดยสาร antioxidant จัะร"ด�วซ์$ ferric ไปเป!น ferrous

FluoresceinPeroxyl radical

antioxidant

Antioxidant activity determination

K. Thaipong et al. / Journal of Food Composition and Analysis 19 (2006) 669–675

TyrosinaseTyrosinase

Tyrosinase inhibitor

Tyrosinase inhibitor

Rate limiting step

Tyrosinase inhibition Assay

Anti tyrosinase Assay

96 well Microplate Assay Format 96 well Microplate Assay Format

Enzyme : Mushroom tyrosinase, Substrate : L-DOPA, L-tyrosinase

OD 475 nm for 20 min OD 475 nm for 20 min

Mixture Mixture

Enzyme

Substrate

Test conpound / Control

Control

Blank

Anti tyrosinase Assay (In melanocyte cell line)

Inhibition of Melanin SynthesisInhibition of Melanin Synthesis

Re

lativ

e m

ela

nin

co

nte

nt

Concentration of test compound

Analysis of tyrosinase activityAnalysis of tyrosinase activity

Re

lativ

e t

yro

sin

ase

act

ivity

Concentration of test compound

Machanism identificationMachanism identification

Morphological Change of Melanocyte Cell Line

Observe the cell morphology the appearance of melanin in a melanosome form (A : control, B : test compounds)

melanosomemelanosome

Anti tyrosinase Assay (In melanocyte cell line)

Anti tyrosinase Assay : Mechanism

microphthalmia-associated transcription factor (MITF)tyrosinase and tyrosinase-related protein 1 (TRP-1)the melanocortin 1 receptor (MC1R)

sample sample

a specific inhibitor of the ERK

pathway

1. Activated extracellular signal-regulated kinase (ERK) pathway Degradation of MITF in transcription factor MITF stimulates TRP-1 gene expression, melanin production

2. Inhibited α-MSH Binding of a-MSH to the MC1R α-MSH controls diverse melanocytic functions such as

proliferation, eumelanin synthesis, and cytokine production activation of the cAMP pathway Increates cAMP increase MITF protein expression

ERK

2. α-MSH

1. TGFR

cAMP

Inhibits Expression of Melanogenesis MarkerInhibits Expression of Melanogenesis Marker

inflammatory acne lesions, NF-κB sign is activated

As a result, inflammatory cytokine genes (eg. TNF-α and IL-1ß) are activated.

TNF-α and IL-1ß will also help to stimulate the proliferation of secondary cytokines, such as IL-8, and also trigger the activation of MAP kinases to stimulate AP-1

AP-1-driven matrix metalloproteinases (MMPs).

Along with collagenase and elastase enzymes brought to the site by (PMNs)

they synthesize collagen and elastin. This is not a perfect solution. Most of the

imperfections could leave clinically untraceable deficits in the forming of the skin layers.

Along with sustained procollagen synthesis acne scarring gets clinically visible

Anti inflammation assay

Nitric oxide

MAP kinases

Nitrite standards

Nitrite standards

Colorimetric Nitric Oxide Assay

A cadmium-copper

(reduction)

Sample

test

Sample

test

Nitrate std.

nitrate standards serially diluted from 200 to 3.13 μM

Standard Curve

Anti inflammatory Assay : machanism

Inhibition of Pro-InflammatoryTNF-α‚ IFN-Υ‚ and IL-1β

Inhibition of Pro-InflammatoryTNF-α‚ IFN-Υ‚ and IL-1β

transcription (RT-PCR)

sampleLPS

Control

sampleLPS

Control

translation (western blot)

Translocation of NFkB p65 Subunit in Nucleus was InhibitedTranslocation of NFkB p65 Subunit in Nucleus was Inhibited

NFkB’s core, the typical transfer factor of inflammation NFkB’s core, the typical transfer factor of inflammation

1. Control2. LPS control3. LPS + sample test 100pg/ml4. LPS + sample test 1ng/ml5. LPS + sample test 10ng/ml

NFfBp65

Actin

western blot.

Anti inflammatory Assay : machanism

Using monoclonal anti-collagen antibodies, is tagged with a fluorescent marker

Collagen synthesis stimulation

Blank

Test compound

Anti-collagen antibodyAnti-collagen antibody Fluorescence

ELISA (Enzyme-Linked ImmunoSorbant Assay) method

Principle : determine if a particular protein (hyaluronic acid and elastin) is present in a fibroblast cell line, treat sample test in 3 different concentration, are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with a antibody to which will bind protein the you want to measure .

Target Ab

Matrix metalloproteinase gene family class of metalloproteinases capable of extracellular matrix (collagen, elastin)

degradation

Such as MMP-2 and MMP-9 (gelatinases A and B), which are the main enzymes able to degrade collagen and other gelatin

Tissue inhibitor of metalloproteinases 1 (TIMP-1) TIMP-1 share the common capability to inactivate metalloproteinase enzymes

Four members of this protein family, TIMP-1, TIMP-2, TIMP-3, and TIMP-4

Gelatinases inhibition

Gelatinases inhibition

The percentage of decrease of MMP and TIMP1 expression, indicate that the sample test can show anti photoaging

Require Products

Hair growth promoter Anti-hair loss Oily & dry hair Anti-dandruff

hair root

The scalp hairroot

shaft

Scalp hair shaft structure

(3 layers)…medulla/cortex/cuticle

Scalp hair root structure

* below the surface of the skin

* enclosed within a hair follicle

* base of hair follicle is the dermal papilla (contains receptors)

cortex cuticle

The structure of the hair (www. Alopecia - hair loss - DolceraWiki.htm)

The hair growth cycle (3 stages )• growth (anagen)

• transitional (catagen)

• resting (telogen)

Selected factors with known hair growth regulatory rolesMcElwee & Sinclair, 2008

Stimulating effect on hair growth

Inhibitory effect on hair growth

Hair-follicle cycling and Signaling molecules controlling hair growth

• androgenetic alopecia (causes from typeII 5α-Reductase)

Causes of hair loss Causes of hair loss Hormone

• Poor nutrition

• Disease

• Medical treatments

• Hair treatments

• Scalp infection

Skin oil (sebum or sebaceous secretions)

• skin micro-organisms

• Individual susceptibility

Non-hormone

Other

• Cicatricial (scarring) alopecia • Alopecia areata• Telogen effluvium

Causes of dandruffCauses of dandruff

Screening test for hair care agents

Hair follicle length determination

Cysteine uptake assay

Cell proliferation

Gene expression & protein determination

5α-Reductase assay (enzymatic activity measurement)

Antimicrobial test

Animal test

Cysteine uptake assay

[35S]cysteine uptake correlates with hair follicle growth

Single follicle from mouse vibrissae tissues were isolated

The follicles were maintained in Williams medium E

Follicles+sample+[35S]cysteine

Radioactivity was measured by liquid scintillation counter

Radioactivity was measured by liquid scintillation counter

Rho et al., 2005

Hair follicle length determination

Hair follicles

Maintained in Dulbecco’s Modified Eagles Medium

Treated with test sample

Hair follicle length was measured under microscope

Adhirajan et al., 2005

Cell proliferation Immortalized human keratinocyte cells (HaCaT) Human dermal papilla cells (DP) Dermal papilla fibroblasts Follicular epithelium

MTT assay Neutral Red assayTrypan blue assay

Cell line

Viable cell + MTT

Formazan crystal

Absorbance measurement at 570 nm

dead cell (porous cell wall) + trypan blue

blue color cell (viable cell was bright & no blue)

Count & calculate

MTT was reduced by viable cell enzyme

Viable cell + neutral red

Viable cell can incorporate and bind the dye in lysosome

Absorbance measurement at 520 nm

Gene expression

Gel Doc

Thermocycler

Electrophoresis

Total RNA

RT-PCR

Real-time PCR

Jang et al., 2007

Rho et al., 2005

RT-PCR result: after transfection of 5αRII into HEK293 cells

Test sample (%)

Western blot analysis -

Protein detection

www.gibthai.com

Protein transfer

DetectionJang et al., 2007

Expression of 5αRII: after transfection of 5αRII gene into HEK293 cells

5α-Reductase assay (enzymatic activity measurement)

Cell lysis

Protein measurement

Mixture: typeII 5α –reductase + cell extract + [3H]testosterone

Steroids extraction

Thin layer chromatography (TLC)

Hyperfilm 3H exposure

Autoradiography visualization

Jang et al., 2007

The result: shows the concentration-dependency of enzymatic activity

Cell (µg) Cell - 5αRII (µg)

Finasteride (%)

Anti-microbial test

Malassezia furfur (Pityrosporum ovale), fungus –cause of dandruff

Spread fungal suspension on a agar plate

Placed the sterile paper discs with test samples on the top of agar

The inhibition zones were observed and recorded

Two-fold dilution of the test sample

Test sample dilution + fungal cell suspension

Calculate as MIC, MBC

Broth dilution methodDisc dilution method

Animal testC57BL mice / C3H mice /

wistar albino rats

The backs of mice were shaved

Agent was applied topically

Observed the darkening on the skin color, histological profiles of skin

or immunohistochemical expression

Control group Treated group

Rho et al., 2005

Harada et al., 2008

Cellulite is a term applied to a skin condition associated with the localized fat deposits

The appearance is frequently described as "orange peel skin" or said to have a “cottage cheese appearance”

Adipocytes (fat cells) are the principle cells implicated in fat storage by adipose tissue

These fat cells contain triglycerides Increased lipolysis of the subcutaneous

adipose means more triglyceride is broken down to lead to smaller fat cells and a reduction of the cellulite appearance

Role of adipocyte in cellulite treatmentRole of adipocyte in cellulite treatment

Inhibition adipogenesis

Preadipocyte(embryo fibroblasts)

Mature adipocyteAdipogenic medium

More lipid contain

Fluorimeter and fluorescent•Excitation 485 nm•Emission 572 nm

+ AdipoRed Assay reagent

Quantification of lipid content Quantification of lipid content

AdipoRed Assay reagent : a solution of the hydrophilic stain Nile Red, is a reagent that enables the quantification of intracellular lipid droplets

Lipolysis Assay

MTS cell viable assay MTS cell viable assay

MTS : 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium