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
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
CORUM 9515 Whitening Mechanism
TRP-2
Three melanin-generating enzymes:
1. 2. 3.
Tyrosinase TRP-2 (DOPAchrome tautomerase) TRP-1 (DHICAoxidase)
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-1
Efficacy Test: In-vitro tyrosinase (I) inhibition Reaction:
Enzyme level
Tyrosine Assay 100
Tyrosine Inhibition Activity IC50
99.8
93.8
100
Inhibition (%)
Inhibition (%)
80 60 37.6
40 20
2.1
9.7
75 5 mg/ml 72.6%
50
7.5 mg/ml 81.4%
10 mg/ml 90.3%
2 mg/ml 51.4%
25 1 mg/ml 37.6%
0 Vit.C
Corum 9515
AA2G
MAP
α-Arbutin
0 0
Sample: 1 mg/ml (0.1%)
4
Concentration (mg/ml)
0.1 % CORUM 9515 can inhibit tyrosinase activity up to 37.6 %. 2 mg/mL of CORUM 9515 can reach 50% inhibition. Ref: Corum Internal Report. Feb 2009.
8
12
Efficacy Test: In-vitro tyrosinase (II) inhibition Reaction:
Enzyme level
DOPA Assay 100
Tyrosine Inhibition Activity IC50
99.1
100
Inhibition (%)
Inhibition (%)
80 54.8
60
31.9
40 22.3 20
2 mg/ml 67.1% 50
8 mg/ml 92.9%
1 mg/ml 47.1% 0.5 mg/ml 40.7%
25
-19.9
0 Vit.C -20
75
4 mg/ml 83.3%
Corum 9515
AA2G
MAP
Sample: 1 mg/ml (0.1%)
α-Arbutin
0 0
5 Concentration (mg/ml)
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.
10
Efficacy Test: In-vitro Tyrosinase, Trp-2 inhibition Protein level
Tyrosinase inhibition
Tyrosinase Level (%)
100
47.5% Inhibition rate
80 60 40
Test: 20
Western blot – Tyrosinase In-vitro melanogenesis inhibition
0 Control 1.
Control 2.
without α-MSH
1 % Corum 9515
1 % AA2G
(with α-MSH)
with α-MSH
Result : 1 % Corum 9515 can inhibit tyrosinase activity up to 47.5 % in protein level.
Ref: Corum Internal Report. Aug 2012.
Efficacy Test: In-vitro Tyrosinase, Trp-2 inhibition Protein level
Trp-2 inhibition 100
Trp-2 Level (%)
80
72.6% Inhibition rate
60
40
Tests:
20
Western blot – Trp-2 In-vitro melanogenesis
0 Control 1. without α-MSH
Control 2.
1 % Corum 9515 with α-MSH
1 % AA2G
inhibition (with α-MSH)
Result : 1 % Corum 9515 can inhibit Trp-2 activity up to 72.6 % in protein level.
Ref: Corum Internal Report. Aug 2012.
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
Efficacy Test: Ferric Reducing Ability
TRP-2
TRP-1
Ref: Briganti, S et al. (2003) Chemical and Instrumental Approaches to Treat Hyperpigmentation Pigment Cell Res 16:101–110.
Efficacy Test: Ferric Reducing Ability pKa value greater - give electron easier - better reducing ability 2’ carbon without ethyl group bonded has better reducing ability
2 1
3
Ascorbic acid (reduced form of Vitamin C)
2
3
2
3
2
3
Ethyl Ascorbic Acid
AA2G
MAP
MW: 204.18
MW: 338.26
MW: 759.22
Efficacy Test: Ferric Reducing Ability e-
Fe 3 + +
100
100
Fe 2+
100
Inhibition (%)
80
60
52.6
40
31.9
20 1 0
Vit.C
Corum 9515
AA2G
Sample: 10 mg/ml (1%) Ref: Corum Internal Report. Feb 2009.
MAP
α-Arbutin
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
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 60
Inhibition (%)
49.75
38.21
40 32.8
20
0 5 mM Kojic acid
15 mg/ml Corum 9515
Ref: “Study of the effect of a test item on depigmentation of cells in culture: melanin assessment”, IDEA, Aug, 2008.
20 mg/ml Corum 9515
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
Efficacy Test: Ex-vivo Melanin Assay MelanoDerm™
by BioInnovation Laboratories, Inc (USA) 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
Efficacy Test: Ex-vivo Melanin Assay Day 0
Day 9
Untreated Tissue
Melanin (μg/tissue)
30
3 % Corum 9515
Melanin Content (μg/tissue) 23.4 16.7
20
10
0
Untreated
3 % Corum 9515
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
Efficacy Test: In-vivo whitening efficacy by SPINCONTROL
Method : Chromametric Analysis
Skin type I
Skin type II
Skin type III
Skin type IV
Ref: Chardon, A., Cretois, I., Hourseau, C., Skin colour typology and sun tanning pathways Int. J. Cosm. Sci, (1991), 13, 191-208
Efficacy Test: In-vivo whitening efficacy by SPINCONTROL
Method: Chromametry (CR-300) 2% Corum 9515 cream
35
Mean Individual Typological Angel
Volunteers: 20 healthy Asian Female 25-40 years old Skin type III
+4.0% 34
33
32
T0
Result: Significant increase on mean ITA
Ref: “In vivo evaluation of the efficacy of one whitening lotion in healthy Asian subjects by chromatography”, Spincontrol Asia, November, 2007.
T +28 Days
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.
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
Mechanism of Photoaging
Efficacy Test:
Anti-inflammation IL-6 test Principle
Stimulate by External Material (e.g. 40 mJ/cm² UVB)
Cytokines Release
Regulate Inflammatory reaction IL6
Corum 9515 100
IL-6 Production (%)
100
100% Inhibition rate
50
18 5
1 0 Control 1
Control 2
1% Corum 9515
0.5% Corum 9515
UVB irradiated Ref: Corum Internal Report.Sep 2012.
0.25% Corum 9515
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
Collagen and its function What is collagen
The main protein of connective tissue Make up 25% - 35% of the whole-body protein content Different types of collagen
Functions
Impart strength, support and skin elasticity
Why important
As skin ages, it produces less collagen and loses its elasticity
Wrinkle formation + Aged skin
Less collagen
Abundant collagen Epidermis
Dermis
The role of ascorbic acid in collagen synthesis Proline Monooxygenase
Proline
Hydroxyproline
makes up collagen
Fe3+
Fe2+
Corum 9515
Inhibition of oxidation (%)
Semidehydroascorbyl radical
Ascorbate
Ferric Reducing Ability 100
100
100
Proline residues convert into hydroxyproline during the synthesis of collagen
80 52.6
60
Hydroxylation requires Vitamin C
31.9
40 20
1
0
Vit C
AA2G
Arbutin
MAP
Corum 9515
Efficacy Test:
Stimulation of natural collagen synthesis by IDEA, France After 48hrs contacts TGF β1 is a very strong collagen synthesis stimulator It is tested with type I collagen 40
30
Stimulation (%)
30
27
20
10
0 10 ng/ml TGF β1
500 ppm Corum 9515
Result: Corum 9515 has a similar effect on collagen synthesis as TGF β1 Ref: “Activity study of a test item on collagen synthesis from human fibroblast culture”, IDEA, June, 2008.
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
Skin penetration profile
Corum 9515 Free-Radical Scavenging Mechanism What are free radicals
Cause
Why important
Normal oxygen atom
Electron lose Create free radical
Atoms, molecules, or ions with unpaired electrons Due to its unpaired electrons, they are often highly reactive Often causes chain reactions
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
Free radical set-off chain reaction
Erosion of cell membrane
Cell membrane
Ref . Stephaniepizzo, (2009). Anti-Up? A Quick View into Antioxidants, Free Radicals, and Your Body
Corum 9515 can neutralize free-radical
Efficacy Test: Radical scavenging test ROS assay after UVB irradiated
Principle
100
100
ROS Production (%)
79.6 64.6 55.1 50
25.6
0
Control 485 nm
Control
5000 ppm 500 ppm 50 ppm Corum 9515 Corum 9515 Corum 9515 UVB irradiated
CORUM 9515 shows great ability on ROS inhibition. 528 nm
Ref: Corum Internal Report. Feb 2013.
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
a. In-vitro, Comet Assay
In-vivo whitening efficacy
b. Ex-vivo
Skin penetration profile
Efficacy Test: DNA Protection Comet Assay
Method: Single Cell Gel Electrophoresis Assay – Comet Assay Theory: Strong alkali A
T
A
A
C
C
T
T
A
T
T
G
G
A
DNA damaged by H2O2
Single cell electrophoresis
-
+ No damage
Serious damage
A A
T
A
T
A
T
T
C G
C
T
G
A
DNA disintegrates
Efficacy Test: DNA Protection Comet Assay
Untreated
Control (H2O2)
Protection rate 25 AA2G
21.9
Corum 9515
AA2G 10,000 ppm
20
C-9515 5,000 ppm
AA2G 5,000 ppm
Protection Rate (%)
C-9515 10,000 ppm
15
10
5
8.9
9.4
4.4
0 5000 ppm
10000 ppm
Result: CORUM 9515 shows high efficiency on DNA protection activity.
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.
Nuclear shrinkage
Nuclear fragmentation
Nuclear fading
Sun burn cells are indicated by arrow.
Efficacy Test: DNA Protection
after UVB irradiation on human living skin explants by BIO-EC, France Explants preparation: From a 45-year-old Caucasian woman 12 skin explants of an average diameter of 11 mm (±1mm) Number of sun burn cell in the epidermis
Spongiosis is slight in the basal layers
Some cells with picnotic nuclei.
*Sunburn cell are indicated by circles
Number of Sunburn Cells/cm
75
53.3 50 35.4 25
0 Control
4% Corum 9515
with UV irradiation
Result: Corum 9515 shows high efficiency on DNA protection activity after UVB irradiation.
CORUM 9515 Efficacy Studies
In-vitro tyrosinase inhibition
Anti-photoaging (Anti-inflammation test)
Reducing activity
Stimulation of collagen synthesis
In-vitro whitening activity
Radical scavenging effect
Ex-vivo melanin assay
DNA protection
In-vivo whitening efficacy
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. ※ Episkin is a skin model as alternatives to animal testing.
Applications: Percutaneous absorption
In vitro skin irritation
In vitro skin corrosion
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 Cumulated permeated amount (μg‧cm-2)
40 35 30 Corum 9515
25 20
AA2G
15
10 5 0 0
1
2
3
4 5 Time (hours)
6
7
8
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
6
Corum 9515
5
AA2G
better in penetration
3.00 Ratio of penertration rate (CORUM 9515 vs AA2G)
Penetration rate (%)
Corum 9515 is 2.4 times
4 3 2 1
2.50 2.00 1.50
1.00 0.50 0.00
0 1
2
3
4
5
6
Time(hours)
7
8
1
2
3
4
5
6
7
Time(hours)
Result: CORUM 9515 shows better skin penetration ability compared with AA2G.
8
Skin Penetration Profile
Human Epidermis Model EPISKIN® :In Vitro Skin Penetration Test Cumulated permeated amount Test materials: 2 % Cream Cumulated permeated amount (μg‧cm-2)
14 12 10 8
Corum 9515
6
AA2G
4 2 0
0
1
2
3
4 5 Time (h)
6
7
8
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)
6
Corum 9515
5
AA2G
Corum 9515 is 5 times
6.00
better in penetration
5.00
Ratio of penertration rate (Corum 9515 vs AA2G)
Penetration rate (%)
Test materials: 2 % Cream
4.00
4
3.00
3
2.00
2
1.00
1 0
0.00 1
2
3
4 5 6 Time(hours)
7
8
1
2
3
4 5 6 Time(hours)
Result: CORUM 9515 shows better skin penetration ability compared with AA2G.
7
8
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: Heat-Stability 0 month 2% 45C without buffer 1 Month 45 ̊C Stability (Transmittance at 440nm ) C-9515
α-Arbutin
AA2G
Vit-C
1 month 2% 45C without buffer
C-9515
α-Arbutin
AA2G
Vit-C
Transmittance %T
100
96.8 90.9
85.1
80 60 41.6 40 20 0 Corum 9515
α-Arbutin
AA2G
Sample: 2%
Ref: Corum Internal Report, November, 2007.
Vit.C
Stability: Crystalline Powder Heat-Stability
99.9
60 Days 45 ̊C Purity
UV detection at 214nm Corum 9515 crystalline powder
99.9
99.9
99.9
99.9
80 Area (%)
HPLC system
100
60 40 20 0 Day 0 Day 1 Day 15 Day 30 Day 60
Result: CORUM 9515 crystalline powder remains stable under 45 ̊C for 60 days
Ref: Corum Internal Report. Feb 2009.
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: CORUM 9515 Photo-Stability 0 month 2% under sunlight without buffer
C-9515
Vit-C
α -Arbutin
AA2G
1 month 2% under sunlight without buffer
C-9515
Ref: Corum Internal Report, November, 2007.
Vit-C
α -Arbutin
AA2G
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 at pH 4 120
Preparation: 2% buffer solution
Area (%)
PURITY assay by HPLC
100 80
A
60
B
40
C
20 0 Day 0 Day 1 Day 7 Day 14 Day 28 Day 56
Day: 56 days at 45 ̊C
Purity at pH 6 120 100 Area (%)
Buffer system: A - Sodium Citrate – Citric Acid B - Na2HPO4- Citric Acid C - NaHCO3 – Citric Acid
80
A
60
B
40
C
20 0 Day 0 Day 1 Day 7 Day 14 Day 28 Day 56
Ref: Corum Internal Report. Feb 2009.
pH Stability under different buffer Stability of pH value in different buffer system (pH 4)
Stability
Preparation: 2% buffer solution
8
pH Value
pH
10
A 6
B
4
C
2
D
0
Day: 56 days at 45 ̊C
Day 1
Day 7 Day 14 Day 28 Day 56
Stability of pH value in different buffer system (pH 6) 10 8
pH Value
Buffer system: A - Sodium Citrate - Citric Acid B - Na2HPO4 - Citric Acid C - NaHCO3 - Citric Acid D -NaOH - Citric acid
Day 0
A
6
B 4
C D
2
0 Day 0 Ref: Corum Internal Report. Feb 2009.
Day 1
Day 7
Day 14
Day 28
Day 56
Anti-oxidant lessen color change 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
C-9515 Sodium bisulfite
Control 0%
2%
2%
2%
0%
0%
0.15%
0.2%
Sodium bisulfite can lessen color change.
But ! Too much sodium bisulfite will accelerate color change.
Minimize Color Change - Chelating Agent Metal Ions promotes oxidation. Chelating agents work to trap and inactivate these metal ions with the formation of chelating complexes in the solution
105 100
Transmittance
95
EDTMP was found to minimize oxidation remarkably in solutions containing Corum 9515 according to our experiments.
90 85
Blank, pH 4.78
80 75
Control, pH 4.78
70
0.1% EDTMP, pH 4.72
65
0.05%EDTMP, pH 4.72
60
0
30
Day
60
90
Clarity of Solution After 90 Days
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!