a case study for ethnic variation of skin conditions among

Transcription

NON-INVASIVE
SKIN EVALUATION METHODS
A CASE STUDY FOR ETHNIC VARIATION OF
SKIN CONDITIONS AMONG ASIAN PEOPLE
DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING
DONGGUK UNIVERSITY,
UNIVERSITY SEOUL,
SEOUL KOREA
CHANG SEO PARK
Contents

Part I : Collaborative Works with CEPP,, UTM
- Searching for Novel Cosmeceuticals from
Tropical Medicinal Herbs

P t II : A Case
Part
C
Study
St d for
f Ethnic
Eth i V
Variation
i ti off Ski
Skin
Conditions Among Asian People
Contents

Part I : Collaborative Works with CEPP,, UTM

P t II : A Case
Part
C
Study
St d for
f Ethnic
Eth i V
Variation
i ti off Ski
Skin
Malaysian
y
Tropical
p
Medicinal Herbs
Labisia pumila (Kacip Fatimah)
- “Queen
Queen of Plants”
Plants for woman health
Eurycoma longifolia (Tongkat Ali)
- Promoting health and enhancing male vitality (pre clinical stage)
Ficus deltoidea (Mas Cotek)
- “True
“T
treasure off nature”” for
f woman hhealth
lh
Smilax calophylla (Ubi Jaga)
- Skin diseases
Mangosteen
- alleviate fever, diarrhea, headaches, and heal wounds
Antiaging
g g Effect of Labisia pumila
p
“Queen of Plants”
Bio-Markers for Cosmeceutical Screening
Ceramides, Transglutaminase
Desquamation related enzymes
NMF
profilaggrin, loricrin etc.
Epidermal
Lamellar body
Homeostasis Proliferation/differentiation markers
MMPs
Histological Structure of DP
DEJ
Laminin,
Type IV & VII collagen
Skin Barrier
Function
Dermall
D
Structure
Collagen I
El ti
Elastin
Proteoglycans
Glycation level of collagen
MMPs
AP1 etc
t
Anti-Inflammatory
y Efficacy
y
80
60
40
20
0.001%
0.004%
0.007%
0.01%
-
+
As 25μM
0
As 12.5μM
M
TNF-α conte
ent
(% o
of the untreated
d control)
100
L bi i pumila
Labisia
il
+ UV (312nm. 25mJ/cm2)
Anti-aging
g g Efficacy
y
25
collagen(μg
g/ml)
20
3 x increase
15
10
5
0.0000
0
0000 5%
%
0.0001 %
0.0005 %
0.001%
+
As 25μM
-
As 12.5μM
A
0
L ab is ia p u m ila
+ U V B (3 12n m . 52m J/c m 2 )
Labisia extract significantly increases collagen production.
Anti-aging
g g Efficacy
y
Gelatin Zymography
≫ Labisia pumila extract reduced the MMP-1 and MMP-9 expression.
Journal of Bioscience and Bioengineering
VOL. 109 No. 3, 291–296, 2010
Ficus deltoidea Has Mutiple Functions
- Anti-aging & Whitening
“True Treasure of Nature”
120
120
100
100
IL-6 content
80
60
40
20
0 .0 05 %
0 .0 08 %
0 .0 1%
(%
% of the untreated c
control)
IL-1α content
(%
% of the untreated control)
Anti-Inflammatory
y Efficacy
y
80
60
40
0 .0
005%
0 .0 0 8 %
0 .0 1 %
20
0
0
-U V
+U V
d ex am eth as on
1 μM
-U V
F IC U S
2
+ U V B (31 2n m , 1 2
2.5
5 m J /cm )
+U V
d e x a m e th a s o n
1μM
F IC U S
+ U V B ( 3 1 2 n m , 1 2 .5
5 m J /c m 2 )
≫ Ficus deltoidea reduced Cox2, IL-1α and IL-6 expression in cultured HaCaT cells.
Anti-aging
g g Efficacy
y
50
MMP-1 conten
nt (μg/ml)
40
30
20
0.001%
0.005%
0.008%
10
0
-UV
UV +UV dexamethason
1μM
FICUS
+ UVB (312nm, 12.5mJ/cm2)
≫ Ficus deltoidea reduced MMP-1 expression and increased type 1 collagen expression.
Anti-Melanogenic(Whitening) Activity of Ficus
500
300
200
100
0.0125 %
0.025 %
0.05 %
01%
0.1
0
- +
Ficus deltoidea
200
150
100
0.0125 %
0.025 %
0.05 %
0.1 %
50
0
Control
(-) (+)
Kojic acid
K
800 μM
Arbutin
2 mM
Control
250
(+) α-MSH 5 nM
- Ficus deltoidea also directly inhibits mushroom tyrosinase
Ficus deltoidea
Arbutin 2 mM
400
Ko
ojic acid 800 μM
Mushrooom tyrosinase activity
( % of th
he untreated control )
Secreeted melanin
( % of the untreated control )
300
(+) Mushroom tyrosinase 10μg/ml
Down Regulation
g
of MITF Expression
p
MITF
(60kDa)
Tyrosinase
(75kDa)
β-actin
(43kDa)
β-actin
(43kDa)
−
+
2mM
800μM 0.0125 0.025
Arbutin Kojic acid
α−MSH 5nM
Ficus
0.05
0.1 (%)
−
+
2mM 800μM 0.0125 0.025 0.05 0.1 (%)
Arbutin Kojic acid
Ficus
α−MSH 5nM
≫ Protein level of tyrosinase and MITF expression was decreased by Ficus deltoidea
≫ Conclusion: Ficus may suppress tyrosinase expression and directly prevents
tyrosinase activity in melanocytes
Regulation
g
of Melanogenesis
g
α MSH
α-MSH
MC1R
St l ffactor
Steel
t
Forskolin, IBMX
cKit
Raf
Ras
cAMP
MEK
U
PKA
hUBC9
CREB
CRE
MITF
U
ERK
MITF-P ser73
MITF-P ser73
MITF degradation
g
MITF
MBOX
Tyrosinase
Melanogenesis
3D Artificial Skin Model Test: MelanoDerm
MelanoDermTM(Matek, USA)
• Co-Culture of Human Keratinocytes
and Melanocytes
Control
• Human Skin-Like Structure
• Reproducible
Kojic
• Ideal for Skin Whitening Studies
• Alternative
Alt
ti tto A
Animal
i l TTesting
ti
Ficus
Archives of Dermatological Research accepted
Mangosteen Leaf Extract Has
S lf Tanning
Self
T
i Activity
A ti it
Mangosteen
g
leaf extract enhances melanogenesis
g
Secreted melanin
(% of th
he untreated conttrol)
1000
300
200
100
800
600
400
200
0
0
Mangosteen Leaves
KA 800uM
FSK 10uM
(-) α-MSH
3.9x10-4%
7.8x10-4%
1.6x10-3%
3.2x10-4%
(+) α-MSH
Control
Mangosteen Sample
(+) α-MSH
Control
(-) α-MSH
Secreted melanin
S
(% of tthe untreated con
ntrol)
400
3.9
7.8
1.6
3.2
+ α-MSH 5 nM
-
+
FSK
10µM
KA
800uM
3.9x10-4 7.8x10-4 1.6x10-3 3.2x10-3(%)
Mangosteen leaves extract
α-MSH 5 nm
x
x
x
x
10-4%
10-4%
10-3%
10-3%
Mangosteen leaf extract stimulates MITF and
T
Tyrosinase
i
gene expression
i
MITF
(60kDa)
Tyrosinase
(70kDa)
ß-actin
(43kDa))
(43kD
-
+
10µM
FSK
800uM
KA
3.9x10-4
Mangosteen leaves extract
α-MSH 5 nm
Archives of Dermatological Research submitted
7.8x10-4 1.6x10-3 3.2x10-3(%)
Summary
y & Future Works




Both Labisia pumila and Ficus deltoides have great
potential to be a novel anti-aging cosmeceutical
Ficus deltoides has additional anti-melanogenic activity and
could be used for a whitening agent
Works for the identification of responsible molecule(s) are
underway
In vivo evidences from human subjects for anti-aging and
whitening efficacies are required
Mariani
Contents

Part I : Collaborative Works with CEPP, UTM

Part II : A Case Study for Ethnic Variation of Skin
In Vivo & In Vitro Methods for Cosmeceuticals
Tests for Anti-Aging Efficacy
• Collagen Assay
• MMP(Collagenase) and Elastase Assay
• Wrinkle measurement : Skin replica
• Elasticity : Cutometry
• Desquamation
• TEWL
Tests for Anti-Aging Efficacy
• Tyrosinase assay
• Melanin Biosynthesis Assay
• MITF expression
• Melanocyte dendricity assay
• Guinea pig skin test
In Vivo Tests for Skin Efficacy
y
Immunohistochemical Staining
Normal
Whitening Assay : Guinea pig
TPA only
Mouse Ear Swelling Test
Griffiths et al. NEJM 1993:329;530-535
No Animal Tests for Cosmetics!
Regulatory context
The Cosmetics Directive foresees a regulatory framework with the aim of phasing out
animal testing. It establishes a prohibition to test finished cosmetic products and cosmetic
ingredients on animals (testing ban), and a prohibition to market in the European
Community, finished cosmetic products and ingredients included in cosmetic products
which were tested on animals ((marketing
g ban).)
The testing ban on finished cosmetic products applies since 11 September 2004; the
testing ban on ingredients or combination of ingredients applies since 11 March 2009.
The marketing ban applies since 11 March 2009 for all human health effects with the
exception of repeated-dose toxicity, reproductive toxicity and toxicokinetics. For these
specific health effects the marketing ban will apply step by step as soon as alternative
methods are validated and adopted in EU legislation with due regard to the OECD
validation process, but with a maximum cut-off date of 10 years after entry into force of
the
h Directive, i.e., 11 March
M h 2013,
20 3 irrespective off the
h availability
l b l off alternative
l
nonanimal tests.
In-vivo Non-Invasive Analysis
y of Living
g Skin
Skin Surface Topography (Wrinkle etc.)
- Replica Image Analysis
- Direct Skin Analyzer(DSA)
Stratum Corneum Hydration
- Skin Surface Conductance – Moisture Content
- Near Infarred Spectrometr
Spectrometry(NIR)
(NIR) – NMF Analysis
Anal sis
- Nuclear Magnetic Resonance(NMR)
Stratum Corneum Barrier Function
- TEWL - Skin Barrier Function
Turnover Cycle of Stratum Corneum ( Epidermal Homeostasis)
- Dansyl chloride method
- Surface area of corneocytes
Ultrasound Imaging
In-vivo Confocal Scanning Laser Microscopy
Efficiency On The Skin
- Surface
S f
evaluation
l ti off living
li i skin,
ki SELS
- Structure of skin
surface is determined
by the following
p
parameters;
;
* Roughness
* Scaling
* Smoothness
* Wrinkle
Ultrasound Measurements
Foundation of CRM



Provide a noninvasive window into living skin for basic and
clinical research.
Skin can be imaged in its native state either in vivo or
freshly biopsied (ex vivo) without the fixing, sectioning, and
staining that is necessary for routine histology.
Dynamic process can be noninvasively observed over an
extended period of time.
Cited from Lasers Med Sci (2007) 22: 73–82
Histology in Verticle Section
Vivascope1500
p
images
g taken on arm
Single captured image
Stratum corneum
Single captured
image
spinous layer
Granular layer
spinous layer with melanin
hat(upper part of papillary)
Granular/spinous layer
spinous layer with papillary
ring of basal cell
Sun-exposed
Sun
exposed skin
Note thinner dermatoglyphs after treatment
(a) Superficial confocal images
((approximately
pp
y 30-µm
µ depth)
p ) of
photodamaged skin at baseline
(b) Superficial confocal images
((approximately
pp
y 30-µm
µ depth)
p ) of
photodamaged skin at 1 month of treatment
Cited from: International Journal of Cosmetic Science, 2008, 30, 1–17
Potential Use of CRM in cosmetic
(a) Confocal images taken at
upper reticular dermis (collagen)
level of a subject volunteer of 41
years old
(b) Confocal images taken at upper
reticular dermis (collagen) level of
a subject volunteer of of 84 years
old
Cited from: International Journal of Cosmetic Science, 2008, 30, 1–17
CRM Images of Papillary Ring
Papillary Ring
Confocal Laser Scanning Microscopy Image
Melanin Hats Protect Nucleus
A PILOT STUDY
COMPARISON OF THREE DIFFERENT ETHNICS :
MALAYSIAN, KOREAN AND VIETNAMESE
Background
g
and Objectives
j
Background
 Several studies indicate that Asian skin maybe more sensitive to exogenous chemicals
probably due to thinner SC and high eccrine gland density
 Asian skin has highest content of ceramide in SC, which corresponds to lowest TEWL
 Asian skin is known, however, to have weakest barrier function against mechanical
challenge, in other words, intercellular cohesion
 Barrier function
f
is also affected
ff
by whole architecture off SC and not just its lipid level
 There is still more to learn and especially about the inherent underlying biological
differences in ethnic skin types
Objectives
 Comparison of skin biophysical properties among Malaysian, Vietnamese, and Korean
 Development of non-invasive
non invasive skin analytical methods by using confocal reflectance
microscopy(CRM)
 Analysis of SC lipid profile of each ethnic group and establishment of methodological
protocols
14
35
12
Male
30
10
Female
Males
Females
25
8
Age (ye
ears)
Nuumber of pa
articipants
Subjects
j
of investigation
g
6
4
20
15
10
2
5
0
Korean
Vietnamese
Malaysian
0
Korean
Vietnamese
Ethnic
Figure 1: Number of participants
Malaysian
Figure 2: Participant’s age in different
gender
d and
d ethnic
th i
Period of staying
y g in Korea
Vietnamese
Malaysian
Male
Female
Male
Female
<6 months
3
5
3
1
6-12 months
4
2
4
3
1 -2 yrs
4
1
4
3
>2yrs
1
0
1
2
Total
13
8
12
9
CONFOCAL REFLECTANCE MICROSCOPY (CRM)
For imaging Human Skin In Vivo and
Measurement of Epidermal Thickness
Confocal Scanning Laser Microcopy
(C f l Reflectance
(Confocal
R fl t
Microscopy
Mi
(CRM))
C t for
Center
f Advanced
Ad
d Colloidal
C ll id l Materials(CACOM),
M t i l (CACOM) Dongguk
D
k University
U i
it
Measurement of Epidermal
Thi k
Thickness
with
ith CRM
Epiderminal
p
margin
g
SET (Short Epidermal Thickness)
LET (Long Epidermal Thickness)
Forearm inner – Male
a) Korean
b) Vietnamese
A
B
A
B
C
D
C
D
Ethnic
c) Malaysian
M
A
B
C
D
Korean
Vietnamese
Malaysian
SET (µm)
33.63 ± 2.89
15.01±2.89
33.08 ± 1.54
LET (µm)
103.44 ± 1.93
75.05±2.87
119.80 ± 5.51
* CRM images of epidermal layers were phtographed by moving the focal plan from the SC surface (A),
down to the top of papillae (B), to the middle of papillae (C), and to the bottom of papillae (D).
Forearm inner – Female
a) Korean
Ethnic
b) Vietnamese
c) Malaysian
M
Korean
Vietnamese
Malaysian
SET (µm)
23.35 ± 0.96
18.35±1.67
18.69 ± 2.42
LET (µm)
93.08 ± 2.73
60.45±1.28
83.53 ± 0.58
* CRM images of epidermal layers were phtographed by moving the focal plan from the SC surface (A),
down to the top of papillae (B), to the middle of papillae (C), and to the bottom of papillae (D).
Epidermal
p
Thickness on The Forearm Inner
100
M a le
F e m a le
Epiderm
mis thicknesss (µm)
Epide
ermal Thickness (μm)
80
60
40
20
0
K o re a n
V ie t
M a la y
ShortThickness
distance
Short Epidermal
S h o r t d is ta n c e
K o re a n
V ie t
M a la y
L o n g d is ta n c e
Long
Long distance
Epidermal Thickness
Significant difference compared with vietnamese at p<0.05
MPA 580
1. Sebumeter, SM815 probe
2 Corneometer,
2.
Corneometer CM825 probe
3. Tewameter, TM300 probe
4 Mexameter,
4.
M
t MX18 probe
b
5. Skin-pH-meter, PH905 probe
6. Reviscometer, RVM600 probe
7. Frictimeter, FR700 probe
8. Sensor for Room Condition probe
Sebumeter,, SM815 probe
p

Determining the Skin Surface Sebum
Forehead,Tzone, Scalp
Cheek,
Eyelid,
Temple
Corner of the
Mouth,Upper
body parts,
B kN k
Back,Neck
Hair
Arms,
Hands,
Legs,
Elb
Elbows
Less
sebum
< 70
< 50
< 40
< 30
0-6
Normal
70-150
50-125
40-90
3070
>6
Oily
> 150
> 125
> 90
> 70
---
Corneometer,, CM825 probe
p
A
Area
Type
Forehead,
Tzone Cheek,
zone,
Cheek
Eyelid, Temple,
Corner of the
Mouth, Upper
body parts, Back,
Neck
Arms,
Hands
Hands,
Legs,
Elbows
V
Very
Dry
D
< 30
< 15
Dry
30-49
15-29
Moisturized
50-59
30-39
Sufficiently
Moisturized
> 60
> 40
Tewameter,, TM300 probe
p
Cylindrical probe head
Hydration(moisture) sensor
Temperature sensor
Transepidermal
p
Waterloss(TEWL)
(
)
Interpretation help
TEWL-values (g/h/㎡)
Very healthy
condition
0-9
Healthy condition
10-14
Normal condition
15-24
Strained condition
25-29
Critical condition
Above 30
* This table is only an approach for the
interpretation of the results
results. It is recommend that
each user should develop an own interpretation
scale.



Transepidermal water loss(TEWL) of the
skin is an important parameter for
evaluating the efficiency function of the
skin water barrier.
more perfect the skin protective coat, the
higher the water content. A low TEWL is
characteristic for a good skin condition.
condition
Dermatologists
g are interested in
normalizing the TEWL.
Mexameter,, MX18 probe
p
Photo
Type
Description
Average Melanin
content
I
Celtic type
0-49
II
Caucasian type
50-99
III
European mixed
type
100-149
IV
Mediterranean
type
150-199
V
Asian/Indian
type
200-299
VI
Black skin type
300-999
 The melanin is measured by specific wavelengths chosen to correspond to different
absorption rates by the pigments.
 For the erythema measurement also specific wavelengths are used, corresponding
to the spectral absorption peak of haemoglobin and to avoid other colour
influences (e. g. bilirubin).
Skin Analysis
SKIN SURFACE MOISTURE, TEWL, MELANIN AND
ERYTHEMA ON THREE DIFFERENT ETHNICS
TEWL
 Forearm
Wateer Loss (g//hr-m2)
25
20
Korean
Vietnamese
Malaysian
15
10
5
0
Male
Female
Significant difference compared with vietnamese at p<0.05
Moisture Content
 Forearm and Face
Watter contennt (arbitra
ary unit)
70
60
50
Korean
Vietnamese
Malaysian
40
30
20
10
0
Male
Female
F
Forearm
Male
Female
F
Face
Melanin Content
Meelanin indeex value ((mJ/cm2)
 Face, Flexor and Extensor Forearm
350
M a le
F e m a le
300
250
200
150
100
50
0
Kor
V ie t
M al
F o r eforearm
a rm
Flexor
In n e r
Kor
V ie t
M al
F o r e aforearm
rm
Extensor
Upper
Kor
V ie t
F Face
ace
M al
Sebum Production
 Face
Sebum vvalue (arbitrary unitt)
400
300
Male
Female
200
100
0
Korean
Vietnamese
Face
Malaysian
CONCLUSION I
•
•
•
•
Data summary;
Thickness : Malaysian > Korean > Vietnamese
TEWL
: Korean > Vietnamese > Malaysian (Skin barrier function)
Moisture, Sebum : Malaysian > Korean > Vietnamese
Melanin : Malaysian > Vietnamese > Korean
There is a good correlation between melanin content measured by
Mexameter
Me
a e e and
a d thee brightness
b g ess of
o papillary
pap a y ring
g observed
obse ved by CRM
C M
Further systematic studies on skin using CRM will provide new options for
non-invasive skin evaluation methods in the future
There is a good correlation between melanin content measured by
Mexameter and the brightness of papillary ring observed by CRM
HUMAN STRATUM CORNEUM LIPID
CHARACTERIZATION AND ETHNIC VARIATIONS
General Structure of Epidermis
p
Denecker et al. J. Cell Biol. 2008:180:451-458
Skin Physiological Lipids & Barrier
F
Function
i
Normal Lipid Barrier
Low
TEWL Value
High
Damaged Lipid Barrier
Stratum
Corneum
1 2
3
1 : CERAMIDE
2 : CHOLESTEROL
3 : FREE FATTY ACID
1. CHANGE OF LIPID COMPOSITION
Epidermis
2. LOSS OF WATER
1.COMPOSITION
1.
COMPOSITION OF SKIN LIPID
- CERAMIDE : > 40%
- CHOLESTEROL : 25%
- FREE FATTY ACID : 25%
*** TEWL : Trans Epidermal Water Loss
- LOWER LEVEL OF CERAMIDE
(LEADS TO SKIN TROUBLE)
DERMIS
- DRY & ROUGH SKIN
- LOSS OF ELASTICITY
- SUSCEPTIBLE TO WRINKLES AND
DISEASES
Factors That May Affect Permeability
B i Function
Barrier
F ti
• Total
amount of the three skin physiologic
lipids, ceramides, cholesterol and fatty acids in SC
• Ratio
R ti among the
th three
th
lipids
li id – 1 : 1 : 1 molar
l ration
ti
eg. Atopic skin – less ceramide
Aged skin – less cholesterol
• Composition of 9 different ceramide classes
• Chain length of fatty acids of both free fatty acids and
fatty acids composed of ceramides
• Number of SC layers
• Thickness
Thi k
off Epidermis
E id
i
• Other protein components for structural integrity of SC
Skin Ceramides
O
O
HN OH
OH
O
Ceramide 1
O
HN OH
OH
Ceramide 3
Ceramide 2
O
HN OH
OH
OH
O
O
HN OH
OH
OH
O
C
Ceramide
id 4
OH
O
HN OH
OH
C
Ceramide
id 6
Ceramide 5
OH
O
HN OH
OH
OH
Ceramide 7
O
HN OH
OH
HO
Hydroxyceramide
y
y
A
OH
O
HN OH
OH
OH
HO
Hydroxyceramide B
Ceramide : N-acylsphingolipids
O
HN OH
OH
OH
Analyses
y
of Skin Barrier Lipids
p
Objectives
• Establish skin
barrier lipid extraction protocols
- Lipid extraction from human subject
• TLC
analysis of skin barrier physiological lipids
- ceramides, cholesterol, and free fatty acids
- relative ratio among three lipid classes
• GC
analysis
y of carbon chain length
g of fatty
y acids
- Long chain fatty acids strengthening barrier function
Methods of Lipid
p Analyses
y

Extraction of SC lipid


Separation
p
of SC lipid
p using
g TLC


The lipid were dissolved in 100 µl chloroform/methanol 2:1 v/v, and 5 µl was
applied to TLC plates and separated using chloroform: acetone:methanol (84:8:8).
The plate were scanned after staining (7.5% Cu-acetate and 2.5% CuSO4 in 8%
H3PO4 (w/w)) and charring at 1600C(2).
C(2)
Preparation of fatty acid methyl ester (FAMEs)


Stratum corneum lipids were extracted from three sites of inner forearm of selected
fifteen subjects by a cup method (technique modified from Serizawa et al. [1]) One
extraction
i with
i h excess cyclohexane/ethanol
l h
/ h l 2:8
2 8 v/v
/ and
d one extraction
i with
i h excess
cyclohexane/ethanol 5:5 v/v. The solvent was then dried up under a flow of
nitrogen.
The fraction of FFAs was scraped off from the TLC plate and extracted with a mixture
of chloroform:methanol (1:1). After evaporation of the solvents, the FFA fraction was
dissolved in 100 μl toluene, saponified in 1N KOH and transmethylated in
BCl3/methanol (14%) using heating block.
Analysis of FAMEs

The FAMEs were separated and analyzed on a GC gas chromatograph.
Skin Barrier Lipid
p Extraction




Stratum corneum lipids were
extracted from four sites of
inner forearm of selected
fifteen subjects by a cup
method (technique modified
from Serizawa et al.
al [1])
Total area of skin extracted
was 10.17cm2
O
One
extraction
t ti
with
ith excess
cyclohexane/ethanol 2:8 v/v
and one extraction with excess
cyclohexane/ethanol 5:5 v/v.
The solvent was then dried up
under a flow of nitrogen.
SC lipid was extracted from four sites of
both inner forearms from each participant
Content of Skin Physiological
y
g
Lipids
p
Ethnic Group
Amount of Lipids Extracted (μg)
Korean
169±45
Vietnamese
171±22
Malaysian
130±16
Total skin area of lipid extraction = 10.17 cm2
HPTLC PLATES AND DEVELOPING
SOLVENT SYSTEM



The lipid were dissolved in 100 µl chloroform/methanol 2:1 v/v, and
5 µl was applied to activated HPTLC plates and separated using chloroform:
chloroform
acetone: methanol (84:8:8).
The plate were scanned after
staining (7.5% Cu-acetate and
2.5% CuSO4 in 8% H3PO4
(w/w)) and charring at 1600C.
STRATUM CORNEUM LIPID ANALYSIS
ONTO TLC PLATE (Silica
(Sili G
Gell 60 F254)
Cholesterol
FFA
Ceramide
SK Chole Std
Flux sterol Mix
Malaysian
Korean
Vietnamese
TLC Analysis of Skin barrier Lipids
- Forearm
Cholesterol
Fatty acids
Vietna
amese
Fatty
y acid
Ceramides
Preparation and Analysis of Fatty
A id Methyl
Acid
M th l Esters
E t



The fraction of FFAs was scraped off from the
plate
p
a e and
a d extracted
e ac ed with
w a mixture
u e of
o
chloroform:methanol (1:1).
After evaporation of the solvents, the FFA
f
fraction
i was di
dissolved
l d iin 100
100μll toluene,
l
saponified in 1N KOH and transmethylated in
BCl3/methanol (14%) using heating block.
The fatty acid methyl esters were analyzed on
a gas chromatography (HP Agilent 6890N GC).
C27:0
C16:0
C26:0
C24
4:1
C23:0
C21:0
0
C18:2
C19:0
C18:0
C22:0
C20:0
C18:2
C24:0
Fatty
y Acid Standard
C27:0
C26:0
C22:0
C20:0
8:2
C18
C18:1
C22:1
C16:0
C24:1
C18:0
FFA fraction of SC lipid
p : Korean
C27:0
C26:0
C22
2:0
C20:0
C18:2
C
C18:1
C22:1
C24:1
C16
6:0
C18:0
p : Vietnamese
C27:0
C26:0
C22:0
C20:0
C18:2 C18:1
C22:1
C16
6:0
C24:1
C18:0
p : Malaysian
y
C27:0
C26:0
C22:0
C20:0
C18:2
C18:1
C22:1
C16:0
C
C24:1
C18:0
FFA Derived From Ceramide : Korean
C27:0
C26:0
C22:0
C20:0
C18:1
C18:2
C22::1
C16:0
1
C24:1
C18:0
FFA Derived From Ceramide : Vietnamese
18:2
C1
C27:0
C26:0
C22
2:0
C20:0
C18:1
C16:0
0
C22:1
C18:0
C24:1
FFA Derived From Ceramide : Malaysian
y
SUMMARY
RT
ID
K1-fa
K2-fa
K3-fa
K4-fa
K5-fa
M1-fa
M2-fa
M3-fa
M4-fa
M5-fa
V1-fa
V2-fa
V3-fa
V4-fa
V5-fa
K1-cer
K2-cer
K3-cer
K4-cer
K5-cer
M1-cer
M2-cer
M3-cer
M4-cer
M5-cer
V1-cer
V2-cer
V3-cer
V4-cer
V5-cer
7.5
16:0
15.70
15.85
18.69
14.99
16.24
15.55
14.23
13.62
16.66
13.69
14.23
14.14
15.89
15.95
18.95
14.03
14.10
16.00
16.18
16.23
12.94
12.50
15.63
13.08
14.75
15.13
13.38
13.29
16.03
15.22
8.1
2.41
2.10
2.05
2.22
3.04
2.44
2.98
2.42
1.92
2.38
2.43
2.59
2.01
2.65
1.71
3.17
2.36
2.61
2.72
2.59
2.89
4.02
3.40
3.72
2.44
3.43
3.78
3.04
2.67
2.64
10.4
2.60
2.20
3.23
1.91
2.43
2.68
2.37
2.14
3.14
2.19
2.86
2.08
2.49
3.75
2.71
2.80
1.90
2.72
4.74
3.96
2.36
2.06
2.73
2.15
3.75
3.85
2.25
2.32
3.77
3.47
1.06
0.00
0.81
1.15
0.79
2.39
0.69
0.71
1.27
0.78
1.07
0.75
0.00
1.11
0.95
1.13
0.65
1.05
0.97
0.93
0.92
1.14
1.05
0.81
1.31
1.36
0.83
0.83
1.12
1.48
10.6
18:2
10 7
10.7
18:1
1 49
1.49
1 11
1.11
1 13
1.13
1 61
1.61
1 04
1.04
4 54
4.54
0 97
0.97
0 92
0.92
1 11
1.11
0 99
0.99
1 14
1.14
1 01
1.01
1 42
1.42
1 22
1.22
1 13
1.13
1 35
1.35
0 82
0.82
1 02
1.02
0 99
0.99
0 94
0.94
1 18
1.18
1 39
1.39
1 23
1.23
1 07
1.07
1 55
1.55
1 49
1.49
0 95
0.95
0 92
0.92
1 23
1.23
1 64
1.64
11.3
18:0
23.25
24.19
26.55
23.50
25.01
23.03
21.53
20.73
24.97
20.80
22.36
21.56
24.08
26.22
33.18
21.55
25.92
26.21
25.75
29.33
17.47
19.95
24.07
19.83
24.17
22.74
22.66
22.08
26.36
23.79
11.8
2.67
1.93
1.91
2.62
3.00
2.86
2.90
3.21
2.04
2.53
3.39
2.97
1.86
2.97
2.67
3.30
3.46
3.03
3.35
3.45
3.34
4.78
3.79
4.56
2.96
4.10
3.95
3.04
2.98
2.91
14.2
0.70
0.43
0.00
0.00
0.00
0.00
1.30
1.23
0.32
0.34
0.54
0.50
0.00
0.00
0.00
0.64
0.00
0.00
0.00
0.33
0.75
0.93
0.44
0.00
0.00
0.00
0.87
0.00
0.00
0.00
14.8
20:0
15.2
0.68
0.60
0.64
1.00
0.58
0.63
0.86
0.82
0.64
0.49
0.90
0.70
0.80
0.98
1.10
0.51
0.77
0.65
0.97
0.72
0.46
0.00
0.67
0.63
0.96
1.03
0.77
0.81
0.99
0.97
0.82
0.98
0.62
1.54
0.73
0.94
1.89
1.42
0.63
0.67
0.57
1.10
1.03
1.43
1.00
1.03
1.04
0.76
1.37
0.91
1.03
1.53
0.70
0.78
1.34
1.34
1.18
1.34
1.44
1.39
16.5
21:0
0.00
0.00
0.00
0.00
0.23
0.00
0.00
0.00
0.00
0.57
0.00
0.44
0.00
1.32
0.64
0.63
0.46
1.09
0.79
0.68
0.50
0.32
0.97
0.88
1.20
1.37
0.86
1.08
1.32
1.37
17.6
22:1
13.38
13.48
11.57
12.81
12.82
11.73
13.56
13.88
11.99
13.50
13.56
13.18
12.68
10.50
8.72
14.28
12.16
11.13
10.15
10.13
15.53
14.13
11.53
13.28
10.49
13.22
13.46
12.46
10.55
10.58
0.98
0.62
1.06
1.93
1.62
1.40
0.96
1.35
1.05
1.47
1.65
1.55
1.76
2.32
1.85
1.40
1.48
1.92
1.88
1.38
1.56
1.40
1.64
1.55
2.08
0.89
1.40
2.11
2.33
2.27
22:0
1.06
0.89
1.08
1.54
1.53
1.35
1.49
1.39
1.28
1.52
1.67
1.56
1.60
2.61
1.95
1.94
1.74
2.05
2.06
2.06
1.83
1.70
2.01
1.66
2.30
2.23
1.70
2.13
2.63
2.55
1.14
0.91
1.02
1.34
0.92
1.36
1.60
1.27
1.20
1.06
1.50
1.24
1.44
1.65
1.19
0.00
0.90
1.28
1.25
0.82
1.23
0.90
1.08
1.05
1.60
0.15
0.95
1.68
1.66
1.79
20.8
24:1
32.07
34.71
29.64
30.73
30.02
27.59
31.62
32.71
31.25
34.81
32.12
33.48
32.30
24.81
20.99
31.63
32.13
28.48
26.80
25.55
35.60
32.30
28.15
34.95
29.09
27.69
31.02
32.87
24.94
27.93
23.7
26:0
0.00
0.00
0.00
1.10
0.00
0.58
0.87
1.06
0.14
1.28
0.00
0.00
0.66
0.00
0.55
0.00
0.12
0.00
0.00
0.00
0.37
0.70
0.31
0.00
0.00
0.00
0.00
0.00
0.00
0.00
25
27:0
0.00
0.00
0.00
0.00
0.00
0.44
0.00
0.00
0.00
0.00
0.00
1.14
0.00
0.00
0.00
0.32
0.00
0.00
0.00
0.00
0.00
0.00
0.15
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.47
0.16
1.13
0.38
0.93
0.00
0.00
0.00
0.52
0.72
0.31
0.00
0.00
0.04
0.00
0.04
0.24
0.42
0.00
0.00
0.00
0.00
0.00
0.00
0.00
18
18.1
18.3
25.8
<C20
50.56
48.42
55.01
49.00
52.12
54.13
47.84
45.79
52.08
44.19
48.92
46.30
48.54
54.85
62.40
48.46
49.98
53.30
55.67
58.49
42.30
46.77
53.01
45.84
51.90
53.13
49.43
46.33
55.14
52.12
≥C20
49.44
51.58
44.99
51.00
47.88
45.87
52.16
54.21
47.92
55.81
51.08
53.70
51.46
45.15
37.60
51.54
50.02
46.70
44.33
41.51
57.70
53.23
46.99
54.16
48.10
46.87
50.57
53.67
44.86
47.88
1.02
0.94
1.22
0.96
1.09
1.18
0.92
0.84
1.09
0.79
0.96
0.86
0.94
1.21
1.66
0.94
1.00
1.14
1.26
1.41
0.73
0.88
1.13
0.85
1.08
1.13
0.98
0.86
1.23
1.09
rℓ=(<C20)/(≥C20)
SFA
17.45
17.35
20.40
18.63
18.58
18.56
17.45
16.88
18.73
17.55
16.80
17.98
18.95
20.86
23.19
17.43
17.19
19.79
20.00
19.69
16.10
15.22
19.75
16.25
19.20
19.75
16.71
17.31
20.97
20.11
MUFA
46.93
49.30
42.34
45.16
43.88
43.85
46.15
47.51
44.35
49.29
46.82
47.66
46.40
36.53
30.83
47.26
45.10
40.63
37.93
36.61
52.32
47.83
40.91
49.30
41.14
42.39
45.43
46.25
36.72
40.15
ru=M/S
2.69
2.84
2.08
2.42
2.36
2.36
2.64
2.81
2.37
2.81
2.79
2.65
2.45
1.75
1.33
2.71
2.62
2.05
1.90
1.86
3.25
3.14
2.07
3.03
2.14
2.15
2.72
2.67
1.75
2.00
Identification of fatty acid composition of human
stratum corneum lipids by comparison with straight
chain fatty acid methyl esters'
C#
Free Fatty acids
Ceramides
Korean
Vietnamese
Malaysian
Korean
Vietnamese
Malaysian
16:0
12.58
12.72
11.65
12.24
11.5
10.68
18:2
0.59
0.63
0.92
0.76
0.88
0.82
18 1
18:1
0 98
0.98
0 95
0.95
1 34
1.34
0 82
0.82
0 98
0.98
1
18:0
18.9
20.51
17.54
20.55
18.53
16.41
20:0
0.54
0.72
0.54
0.58
0.72
0.42
21:0
0.04
0.39
0.09
0.58
0.95
0.60
22:1
9.87
9.33
10.21
9.26
9.43
10.02
22:0
0
0.94
0
9
1.51
5
1.11
1.57
57
1.77
77
1.47
7
24:0
24.21
22.86
24.94
23.1
22.65
24.71
Highest amount
low amount
lowest amount
CONCLUSION II
•
•
•
•
•
The lowest skin barrier function of Malaysian volunteers seemed to be due
to lesser amount of total SC lipids compared to those of Korean and
Vietmanese.
Vietmanese
Relative ratio of three major SC lipid classes ceramide, cholesterol, and
fatty acids did not show any significant difference among Malaysian,
Korean, and Vietnamese.
The chain length profiles of fatty acids showed similar pattern among three
ethnic groups.
groups
There is still more to learn and especially about the inherent underlying
biological differences in ethnic skin types especially.
Future works will focus on the large scale analyses of lipids profile and
content among three major SC lipids, ceramides, cholesterol and fatty acids.
THANK YOU
FOR YOUR ATTENTION

a case study for ethnic variation of skin conditions among

Transcription

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