LOGO Screening test for Development of Cosmeceuticals
LOGO
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