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New Generation Vitamin C Derivative
CORUM 9515 3-O-Ethyl Ascorbic Acid
AS POTENT AS VITAMIN C
WITH BETTER STABILITY
CORUM 9515 INCI: 3- O – Ethyl ascorbic acid
Also known as:
3- O – ethyl ascorbic ether
3- O – ethyl ascorbic acid
Physical Properties
Purity > 99.0%
White crystalline powder
Water soluble
Metabolized by the human body in the same
manner as L- ascorbic acid (Vitamin C)
Excellent stability
CAS: 86404-04-8
MW: 204.18
The Functions of CORUM 9515
Skin lightening & Balance skin tone
Reduce dark spot & age spot
Anti-photoaging
Collagen synthesis
Reversing auto-oxidation
Radical scavenging
DNA protection
Easy to penetrate the epidermis
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
CORUM 9515 Whitening Mechanism
Inhibit the reaction center Cu+2 ion of tyrosinase
Inhibit the activity of TRP-2
Reduce oxidized melanin pigments
Ref: Briganti, S et al. (2003) Chemical and Instrumental Approaches to Treat
Hyperpigmentation Pigment Cell Res 16:101–110.
TRP-2
TRP-1
Three melanin-generating enzymes:
1. Tyrosinase
2. TRP-2 (DOPAchrome tautomerase)
3. TRP-1 (DHICAoxidase)
99.8
37.6
2.1 9.7
93.8
0
20
40
60
80
100
Vit.C Corum
9515
AA2G MAP α-Arbutin
Sample: 1 mg/ml (0.1%)
Tyrosine Assay
Inh
ibitio
n (
%)
Efficacy Test: In-vitro tyrosinase (I) inhibition
0.1 % CORUM 9515 can inhibit tyrosinase activity up to 37.6 %.
2 mg/mL of CORUM 9515 can reach 50% inhibition.
Reaction:
Ref: Corum Internal Report. Feb 2009.
0
25
50
75
100
0 4 8 12
Concentration (mg/ml)
5 mg/ml
72.6%
7.5 mg/ml
81.4%
10 mg/ml
90.3%
Tyrosine Inhibition Activity IC50
2 mg/ml
51.4%
1 mg/ml
37.6%
Enzyme level
Inh
ibitio
n (
%)
Efficacy Test: In-vitro tyrosinase (II) inhibition
0.1 % Corum 9515 can inhibit tyrosinase activity up to 54.8%.
1 mg/mL of Corum 9515 can reach 50% inhibition.
Ref: Corum Internal Report. Feb 2009.
99.1
54.8
22.3
31.9
-19.9
-20
0
20
40
60
80
100
Vit.C Corum
9515
AA2G MAP α-Arbutin
Sample: 1 mg/ml (0.1%)
0
25
50
75
100
0 5 10
Concentration (mg/ml)
2 mg/ml
67.1%
4 mg/ml
83.3%
8 mg/ml
92.9%
DOPA Assay Tyrosine Inhibition Activity IC50
1 mg/ml 47.1%
0.5 mg/ml
40.7%
Enzyme level Reaction:
Inh
ibitio
n (
%)
Inh
ibitio
n (
%)
Tyrosinase inhibition
Result : 1 % Corum 9515 can inhibit tyrosinase activity up to 47.5 % in protein level.
Test:
Western blot – Tyrosinase
In-vitro melanogenesis inhibition
(with α-MSH)
Ref: Corum Internal Report. Aug 2012.
0
20
40
60
80
100
Control 1. Control 2. 1 % Corum 9515 1 % AA2G
without α-MSH with α-MSH
47.5%
Inhibition
rate
Protein level
Tyro
sin
ase
Le
ve
l (
%)
Efficacy Test: In-vitro Tyrosinase, Trp-2 inhibition
Trp-2 inhibition
Result : 1 % Corum 9515 can inhibit Trp-2 activity up to 72.6 % in protein level.
Ref: Corum Internal Report. Aug 2012.
0
20
40
60
80
100
Control 1. Control 2. 1 % Corum 9515 1 % AA2G
with α-MSH without α-MSH
72.6%
Inhibition
rate
Protein level
Tests:
Western blot – Trp-2
In-vitro melanogenesis
inhibition (with α-MSH)
Trp
-2 L
ev
el (
%)
Efficacy Test: In-vitro Tyrosinase, Trp-2 inhibition
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Ref: Briganti, S et al. (2003) Chemical and Instrumental Approaches to Treat
Hyperpigmentation Pigment Cell Res 16:101–110.
TRP-2
TRP-1
Efficacy Test: Ferric Reducing Ability
1
2 3
2 3
pKa value greater - give electron easier
- better reducing ability
2’ carbon without ethyl group bonded
has better reducing ability
Ascorbic acid (reduced form of Vitamin C)
Ethyl Ascorbic Acid AA2G MAP
Efficacy Test: Ferric Reducing Ability
2 3 3 2
MW: 204.18 MW: 338.26 MW: 759.22
Fe 3 + + Fe 2+ e-
Ref: Corum Internal Report. Feb 2009.
100 100
52.6
1
31.9
0
20
40
60
80
100
Vit.C Corum 9515 AA2G MAP α-Arbutin
Sample: 10 mg/ml (1%)
Inh
ibitio
n (
%)
Efficacy Test: Ferric Reducing Ability
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Efficacy Test:
In-vitro whitening activity study - melanin assessment
by IDEA
Method: 12 hours contact with the product and
stimulation during 48 hours with theophylline at 0.5 mM.
Result: 49.75% whitening effect, 20mg/ml Corum 9515
Ref: “Study of the effect of a test item on depigmentation of cells in culture: melanin
assessment”, IDEA, Aug, 2008.
32.8
38.21
49.75
0
20
40
60
5 mM Kojic acid 15 mg/ml Corum 9515 20 mg/ml Corum 9515
Inh
ibitio
n (
%)
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Efficacy Test: Ex-vivo Melanin Assay MelanoDerm™
Purpose
using an in-vitro tissue model of the human epidermis prepared from cultured
human keratinocytes and melanocytes.
both water-soluble and water-insoluble materials
skin darkening agents or skin lightening agents
by BioInnovation Laboratories, Inc (USA)
Untreated Tissue 3 % Corum 9515
Efficacy Test: Ex-vivo Melanin Assay
23.4
16.7
0
10
20
30
Untreated 3 % Corum 9515
Me
lan
in (
μg
/tis
sue
)
Melanin Content (μg/tissue)
Day 0 Day 9
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
by SPINCONTROL
Method : Chromametric Analysis
Efficacy Test: In-vivo whitening efficacy
Skin type IV
Skin type III
Skin type II
Skin type I
Ref: Chardon, A., Cretois, I., Hourseau, C., Skin colour typology and sun tanning
pathways Int. J. Cosm. Sci, (1991), 13, 191-208
Volunteers:
20 healthy Asian
Female
25-40 years old
Skin type III
Method:
Chromametry (CR-300)
2% Corum 9515 cream
Ref: “In vivo evaluation of the efficacy of one whitening lotion in healthy Asian subjects by
chromatography”, Spincontrol Asia, November, 2007.
32
33
34
35
T 0 T +28 Days
Me
an
In
div
idu
al Ty
po
log
ica
l
An
ge
l
+4.0%
Result: Significant increase on mean ITA
by SPINCONTROL
Efficacy Test: In-vivo whitening efficacy
Ref: “In vivo evaluation of the efficacy of one whitening lotion in healthy Asian subjects by
chromatography”, Spincontrol Asia, November, 2007.
Efficacy Test: In-vivo whitening efficacy
Ref: “In vivo evaluation of the efficacy of one whitening lotion in healthy Asian subjects by
chromatography”, Spincontrol Asia, November, 2007.
Efficacy Test: In-vivo whitening efficacy
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Mechanism of Photoaging
Efficacy Test: Anti-inflammation IL-6 test
Ref: Corum Internal Report.Sep 2012.
Principle Stimulate by External Material (e.g. 40 mJ/cm² UVB) Cytokines Release
IL6
Regulate Inflammatory reaction
Corum 9515
1
100
5
18
0
50
100
Control 1 Control 2 1% Corum
9515
0.5% Corum
9515
0.25% Corum
9515
IL-6
Pro
du
ctio
n (
%)
UVB irradiated
100%
Inhibition
rate
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Collagen and its function
The main protein of connective tissue Make up 25% - 35% of the whole-body protein content Different types of collagen
Impart strength, support and skin elasticity
What is
collagen
Functions
Why
important As skin ages, it produces less collagen and loses its elasticity
Wrinkle formation + Aged skin
Epidermis
Dermis
Abundant collagen Less collagen
Fe3+ Fe2+
Proline Hydroxy-
proline
Proline Monooxygenase
Semidehydroascorbyl
radical
Ascorbate
makes up collagen
The role of ascorbic acid in collagen synthesis
100
52.6
31.9
1
100
0
20
40
60
80
100
Vit C AA2G Arbutin MAP Corum
9515
Inh
ibitio
n o
f
ox
ida
tio
n (%
)
Ferric Reducing Ability
Corum 9515
Proline residues convert into
hydroxyproline during the
synthesis of collagen
Hydroxylation requires
Vitamin C
by IDEA, France
After 48hrs contacts
TGF β1 is a very strong collagen synthesis stimulator It is tested with type I collagen
Ref: “Activity study of a test item on collagen synthesis from human fibroblast culture”, IDEA, June, 2008.
Result: Corum 9515 has a similar effect on collagen synthesis as TGF β1
30 27
0
10
20
30
40
10 ng/ml TGF β1 500 ppm Corum 9515
Stim
ula
tio
n (
%)
Efficacy Test: Stimulation of natural collagen synthesis
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Corum 9515 Free-Radical Scavenging Mechanism
Atoms, molecules, or ions with unpaired electrons Due to its unpaired electrons, they are often highly reactive
Often causes chain reactions
What are free
radicals
Cause
Why
important
UV rays, stress & environmental pollution etc.
It can participate in unwanted side reactions resulting in cell
damage Many form of cancer are thought to be the result of reactions
between free radicals and DNA
Cell membrane
Free radical set-off chain reaction
Normal oxygen atom
Electron lose Create free radical
Erosion of cell membrane
Corum 9515 can neutralize free-radical
Ref . Stephaniepizzo, (2009). Anti-Up? A Quick View into Antioxidants, Free Radicals, and Your Body
Efficacy Test: Radical scavenging test ROS assay after UVB irradiated
Ref: Corum Internal Report. Feb 2013.
25.6
100
55.1
64.6
79.6
0
50
100
Control Control 5000 ppm
Corum 9515
500 ppm
Corum 9515
50 ppm
Corum 9515
RO
S P
rod
uc
tio
n (
%)
UVB irradiated
Principle
CORUM 9515 shows great ability on ROS inhibition.
485 nm
528 nm
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
a. In-vitro, Comet Assay
b. Ex-vivo
Skin penetration profile
Efficacy Test: DNA Protection Comet Assay
Method: Single Cell Gel Electrophoresis Assay – Comet Assay
Theory:
A T A C T A C
T A T G A T G T A T G A T G
A T A C T
A C
DNA damaged
by H2O2
Strong alkali
DNA disintegrates
Single cell electrophoresis
No damage
Serious damage
+ -
Efficacy Test: DNA Protection Comet Assay
C-9515 5,000 ppm
AA2G 10,000 ppm AA2G 5,000 ppm
Control (H2O2) Untreated
C-9515 10,000 ppm
4.4
9.4 8.9
21.9
0
5
10
15
20
25
5000 ppm 10000 ppm
Pro
tec
tio
n R
ate
(%
)
AA2G
Corum 9515
Result: CORUM 9515 shows high efficiency on DNA protection activity.
Protection rate
Nuclear fading
Nuclear shrinkage
Nuclear fragmentation
What are Sun Burn Cells?
Sunburn cells (SBC) are keratinocytes
undergoing apoptosis as a protective
mechanism against the carcinogenic effects of UVB irradiation which irreversibly damages
their DNA or other chromophores.
Sun burn cells are indicated by arrow.
Efficacy Test: DNA Protection after UVB irradiation on human living skin explants
*Sunburn cell are
indicated by circles
Number of sun burn cell in the epidermis
Result: Corum 9515 shows high efficiency on DNA protection activity after UVB irradiation.
by BIO-EC, France
53.3
35.4
0
25
50
75
Control 4% Corum 9515
Nu
mb
er
of
Su
nb
urn
Ce
lls/
cm
with UV irradiation
Spongiosis is slight in the basal layers
Some cells with picnotic
nuclei.
Explants preparation:
From a 45-year-old Caucasian woman 12 skin explants of an average diameter
of 11 mm (±1mm)
In-vitro tyrosinase inhibition
Reducing activity
In-vitro whitening activity
Ex-vivo melanin assay
In-vivo whitening efficacy
CORUM 9515 Efficacy Studies
Anti-photoaging
(Anti-inflammation test)
Stimulation of collagen synthesis
Radical scavenging effect
DNA protection
Skin penetration profile
Skin Penetration Profile Human Epidermis Model EPISKIN®:In Vitro Skin Penetration Test
in vitro reconstructed human epidermis which is similar to the in vivo human
epidermis.
Applications:
Percutaneous absorption
In vitro skin corrosion
※ Episkin is a skin model as alternatives to animal testing.
In vitro skin irritation
Pharmacology /Toxicology
Test materials:
2 % solution
2 % Whitening cream
Skin Penetration Profile Human Epidermis Model EPISKIN® :In Vitro Skin Penetration Test
Cumulated permeated amount
Test materials: 2 % solution
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8
Time (hours)
Corum 9515
AA2G
Cu
mu
late
d p
erm
ea
ted
am
ou
nt
(μg‧
cm
-2)
Result: CORUM 9515 shows better skin penetration ability compared with AA2G.
Skin Penetration Profile Human Epidermis Model EPISKIN® :In Vitro Skin Penetration Test
Penetration rate ‧Ratio (Corum 9515 vs. AA2G)
Test materials: 2 % solution
Result: CORUM 9515 shows better skin penetration ability compared with AA2G.
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8
Pe
net
rati
on
rat
e (
%)
Time(hours)
Corum 9515
AA2G
0.00
0.50
1.00
1.50
2.00
2.50
3.00
1 2 3 4 5 6 7 8
Rat
io o
f p
en
ert
rati
on
rat
e
(C
OR
UM
95
15
vs
AA
2G
)
Time(hours)
Corum 9515
is 2.4 times better in
penetration
Result: CORUM 9515 shows better skin penetration ability compared with AA2G.
Skin Penetration Profile Human Epidermis Model EPISKIN®:In Vitro Skin Penetration Test
Cumulated permeated amount
Test materials: 2 % Cream
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8
Time (h)
Corum 9515
AA2G
Cu
mu
late
d p
erm
ea
ted
am
ou
nt
(μg‧
cm
-2)
Skin Penetration Profile Human Epidermis Model EPISKIN® :In Vitro Skin Penetration Test
Penetration rate ‧Ratio (Corum 9515 vs. AA2G)
Test materials: 2 % Cream
Result: CORUM 9515 shows better skin penetration ability compared with AA2G.
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8
Pe
net
rati
on
rat
e (
%)
Time(hours)
Corum 9515
AA2G
0.00
1.00
2.00
3.00
4.00
5.00
6.00
1 2 3 4 5 6 7 8
Rat
io o
f p
en
ert
rati
on
rat
e
(C
oru
m 9
51
5 v
s A
A2
G)
Time(hours)
Corum 9515
is 5 times better in
penetration
Stability Tests
Heat-stability (45 ̊C , 1 month)
- color (Transmittance in 440nm )
- purity (HPLC)
Photo-stability (sunlight, 1 month)
Buffer system and pH effect (45 ̊C, 56 days and RT, 90 days)
AA2G C-9515
0 month 2% 45C without buffer
Stability: Heat-Stability
1 month 2% 45C without buffer
Vit-C α-Arbutin
Ref: Corum Internal Report, November, 2007.
1 Month 45 ̊C Stability
(Transmittance at 440nm )
96.8 90.9
85.1
41.6
0
20
40
60
80
100
Corum 9515 α-Arbutin AA2G Vit.C
Tra
nsm
itta
nc
e %
T
Sample: 2%
AA2G C-9515 Vit-C α-Arbutin
Stability: Crystalline Powder Heat-Stability
60 Days 45 ̊C Purity
HPLC system
UV detection at 214nm
Corum 9515 crystalline powder
Result: CORUM 9515 crystalline powder remains stable under 45 ̊C for 60 days
Ref: Corum Internal Report. Feb 2009.
99.9 99.9 99.9 99.9 99.9
0
20
40
60
80
100
Day 0 Day 1 Day 15 Day 30 Day 60
Are
a (
%)
Stability Tests
Heat-stability (45 ̊C , 1 month)
- color (Transmittance in 440nm )
- purity (HPLC)
Photo-stability (sunlight, 1 month)
Buffer system and pH effect (45 ̊C, 56 days and RT, 90 days)
C-9515 α -Arbutin Vit-C AA2G
Stability: CORUM 9515 Photo-Stability
C-9515 α -Arbutin Vit-C AA2G
0 month 2% under sunlight without buffer
1 month 2% under sunlight without buffer
Ref: Corum Internal Report, November, 2007.
Stability Tests
Heat-stability (45 ̊C , 1 month)
- color (Transmittance in 440nm )
- purity (HPLC)
Photo-stability (sunlight, 1 month)
Buffer system and pH effect (45 ̊C, 56 days and RT, 90 days)
Stability under different pH & buffer
PURITY assay by HPLC
Preparation:
2% buffer solution
Day: 56 days at 45 ̊C
Buffer system:
A - Sodium Citrate – Citric Acid
B - Na2HPO4- Citric Acid
C - NaHCO3 – Citric Acid
Ref: Corum Internal Report. Feb 2009.
0
20
40
60
80
100
120
Day 0 Day 1 Day 7 Day 14 Day 28 Day 56
Are
a (%
)
Purity at pH 4
A
B
C
0
20
40
60
80
100
120
Day 0 Day 1 Day 7 Day 14 Day 28 Day 56
Are
a (%
)
Purity at pH 6
A
B
C
pH Stability under different buffer
pH Stability
Preparation:
2% buffer solution
Day: 56 days at 45 ̊C
Buffer system:
A - Sodium Citrate - Citric Acid
B - Na2HPO4 - Citric Acid
C - NaHCO3 - Citric Acid
D -NaOH - Citric acid
0
2
4
6
8
10
Day 0 Day 1 Day 7 Day 14 Day 28 Day 56
pH
Va
lue
Stability of pH value in different buffer system (pH 4)
A
B
C
D
0
2
4
6
8
10
Day 0 Day 1 Day 7 Day 14 Day 28 Day 56
pH
Va
lue
Stability of pH value in different buffer system (pH 6)
A
B
C
D
Ref: Corum Internal Report. Feb 2009.
Corum 9515 Ethyl Ascorbic Acid is a powerful anti-oxidant
Thus a stronger Anti-oxidant is recommended to be used in formulation prevent losing Corum 9515’s effect before it
reaches its target area
Anti-oxidant
Corum 9515
(Ethyl Ascorbic Acid)
Other ingredients
Anti-oxidant lessen color change
45 ̊C for 6 months
2% 0%
2%
0.15%
2%
0.2%
C-9515
Sodium bisulfite
Sodium bisulfite can lessen color change.
But ! Too much sodium bisulfite will accelerate color change.
0% 0%
Control
Anti-oxidant lessen color change
60
65
70
75
80
85
90
95
100
105
0 30 60 90
Tra
nsm
itta
nce
Day
Blank, pH 4.78
Control, pH 4.78
0.1% EDTMP, pH 4.72
0.05%EDTMP, pH 4.72
Minimize Color Change - Chelating Agent
EDTMP was found to minimize oxidation remarkably in
solutions containing Corum
9515 according to our
experiments. Clarity of Solution After 90 Days
Metal Ions promotes oxidation. Chelating agents work to trap and inactivate these metal ions with the formation of chelating complexes
in the solution
CORUM 9515 Toxicological Information
Skin Irritancy Test by IDEA, France
2% CORUM 9515 on the external face of the arm maintained over 48 hours with the
help of a semi-occlusive patch.
CORUM 9515 is found to be non-irritant after 48 hours semi-occlusive patch test.
Cytotoxicity Test by Evic, France Cytotoxicity test on CORUM 9515 diluted with 10% distilled water.
CORUM 9515 diluted at 10 % with distilled water was judged negligible.
AMES Test by Vivotecnia, Spain CORUM 9515 were found to be non mutagenic and non pro-mutagenic.
CORUM 9515 Summary
Effective and stable skin lightening agent
Balance the skin tone
Reduce dark spot
Prevent photoaging
Increase collagen synthesis
Excellent anti-oxidation properties
Scavenge radical
DNA protection
Thank you for your attention!