Sytenol® A: An Effective

“Retinol-Like” Functional Anti-Aging Ingredient

Sytheon Ltd., Boonton, NJ 07005

Tel: 973-988-1075
E-mail: info@sytheonltd.com
Website: www@sytheonltd.com
Sytenol® A vs. Retinol: A Better Choice

 Retinol has been demonstrated to be very effective in:

1. Reversing aging
2. Protecting skin from further damage and
3. Improving problem skin
 But, Retinol has numerous problems:
1. Side effects
 Skin irritation, skin sensitization and teratogenicity
2. Photochemical and hydrolytic instability
 use primarily restricted to night time
3. Incompatibility
• difficulty in formulating products
 Sytenol A is equally effective in addressing all three skin care issues and is:
®

o Devoid of safety issues

o Compatible with a wide variety of cosmetic ingredients
o Easy to formulate
o Devoid of instability problem
o All day topical application possible

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What is Sytenol® A?

Babchi seeds
Trade name Sytenol A
INCI name Bakuchiol
Chemical Phenol, 4-[1E, 3S)-3-ethenyl-3,
name 7-dimethyl-1, 6-octadienyl
H3C
CAS # 10309-37-2
Origin Natural; from edible seeds;
Ayurvedic
Appearance Pale yellow viscous liquid
Purity  90%
H3C CH3 OH
Stability Excellent photochemical &
hydrolytic stability
λmax 261 nm
Patent status US 8,529,967; multiple US and
international pending patents

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Is there any Structural Resemblance Between
Sytenol A and Retinoids?

H3C

H3C CH3 OH

Sytenol® A

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What is Aging Skin?

 As we age, skin becomes thin, dull and dry with more visible fine lines,
wrinkles and age spots because of
• Epidermal and dermal thinning
• Decreased number of keratinocytes and fibroblasts
• Reduction in the amount and organization of connective tissue
• Flattening of epidermal-dermal junction
 Skin aging entails drastic changes in both the
• Extracellular matrix (ECM) and more specifically
• Dermal-epidermal junction (DEJ)
 Retinoid treatment does, in fact, have the capacity to reverse (at least some
of) the cellular and biochemical events that underlie in chronological and
photo-damaged skin

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How to Define & Portray Molecular Signature of Retinol
and a “Retinol-like” Compound?

 A comparative microarray experiments have been carried out side by side with Retinol
and Sytenol® A under identical condition to define the molecular signature of the two
compounds.
 Volcano plot is a type of scatter plot that is used to quickly identify meaningful
changes in large datasets, such as data from DNA microarray analysis. It plots significance
versus fold-change on the y- and x-axes, respectively.
• A volcano plot is constructed by plotting the negative log of the p-value on the y-axis (usually base
10). This results in data points with low p-values (highly significant) appearing towards the top of
the plot. For example, if p value of 0.05 is set as the threshold for statistical significance, all point
situated above the value of 1.3 on the y axis are statistically significant. The x-axis is the log of the
fold change between the two conditions. For example, if 2 fold change is set as threshold, all
points to the right of 1 and to the left of -1 on the x axis are of interest.

• In summary, plotting points in this way results in two regions of interest in the plot: those points
that are found towards the top of the plot and that are far to either the left- or the right-hand
side. These represent values that display large magnitude fold changes (hence being left- or right-
of center) as well as high statistical significance (hence being towards the top).
Conclusion
• Comparative Volcano plots of Sytenol® A and Retinol show dramatic similarities in gene
expression profiles of the two compounds indicating similar functional analogy.

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Sytenol A vs Retinol: Comparative Gene Expression
Profile Demonstrates “Retinol-like” Functional Attributes of Sytenol A
DNA microarray study done using full thickness Epiderm tissue from Mattek

Volcano Plots

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Sytenol A provides Retinol-like Activity by Modulating
Key Retinoid Binding & Metabolizing Genes: Comparative Gene
Expression Profile

DNA microarray: Used full thickness

Epiderm tissue from Mattek;
Considered expression profile of Threshold of stimulatory activity Threshold of inhibitory activity
over  2-fold change with P < 0.05

Gene expression profile: Fold-change vs Control

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Sytenol A vs Retinol: Comparative Modulation of Key
Dermal-Epidermal Junction Genes

Threshold of stimulatory activity

An intact basement membrane at

the epidermal-dermal junction is
essential in maintaining youthful
skin.
As we age, flattening of the DEJ
causes dramatic loss of
"undulating shape”
(responsible for skin tightening)

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Sytenol A vs Retinol: Comparative Modulation of Key
Extracellular Matrix Genes

Threshold of stimulatory activity

An intact & hydrated

extracellular matrix protein
levels are essential in
maintaining youthful skin

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Sytenol A vs Retinol: Comparative Modulation of
Cytoskeleton genes – Microtubules, Microfilaments &
Intermediate Filaments
The Cytoskeleton gives the Cell its Structure, Form, Elasticity & Mobility.

Threshold of stimulatory activity

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Sytenol A vs Retinol: Comparative Modulation of
Cytoskeleton Genes – Intermediate Filaments

Intermediate filaments maintain cell’s shape providing mechanical support, preventing

excessive stretching, and supporting other organelles

Threshold of stimulatory
activity

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Comparable & Beneficial Expression Profile seen with
Sytenol A and Retinol on Key Dermally Relevant Genes

 Cell adhesion molecules (CAMs)

• Responsible for cell-cell communication
• Proteins located on the cell surface and are involved with the binding
with other cells or with the extracellular matrix (ECM) in the process,
called cell adhesion
 Tight junctions (TJ)
• Responsible barrier homeostasis
• Barrier forming cell–cell junctions, especially for inside–out barrier.
 Epidermal differentiation
• Essential for maintaining barrier function
• Keratinocytes undergo a complex, highly organized and tightly controlled
differentiation program leading to cornification and finally to
desquamation.

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Confirmation of DNA Microarray Study Results by rtPCR
Studies

 Out of 18 genes (LRAT, TIG-1, LAM3, ITGB4, COL4A6, COL1A2, CTHRC1, HAS3, PI3, KRT19, F11R,
) selected, 16 genes modulated
FGFBP1, ACTN4, NFKB1, PARP12, CLDN7, CDH1, SIR2

similarly by both Retinol and Sytenol® A

 13 were significantly up-regulated
• This up-regulation was consistent with the DNA microarray results for 12 of these
genes.
• One gene, which was found to be down-regulated in DNA microarrays, but up-
regulated in rtPCR was NFKB1.
 In summary, rtPCR test confirmed the functional analogy of
Retinol and Sytenol® A
• Results validated stimulatory effects of Sytenol® A on several genes, whose
expression is beneficial for skin integrity and function

rtPCR - reverse transcriptase polymerase chain reaction: a sensitive indirect method for measuring the expression
of a protein in a cell or tissue, by analyzing the level of the messenger RNA encoding the protein

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Sytenol A Stimulates collagens Type I and IV
(Cell Culture Study)

Sytenol® A and Retinol: Increased collagen output per cell without increasing the cell numbers
Collagen I: % Stimulatory effects vs control Collagen IV: % Stimulatory effects vs control

160 160

120 120

80 80

40 40

0 0
Control Sytenol A Retinol MAP Control Sytenol A Retinol MAP

Quantification of collagenic proteins: type I and IV

•Cell Line – Normal human dermal fibroblasts; No cell proliferation seen with Sytenol® A & Retinol at 10 µg/ml; 40% cell
proliferation seen with MAP at 10 µg/ml
•Cell Culture - DMEM with 5% calf serum
•Use level – 10 µg/ml; dissolved in DMSO & then diluted in water; Water used as a control
•Protocol - Tested for type I & IV collagen by sandwich ELISA using affinity-purified antibodies, followed by streptavidin-
avidin-HRP conjugate and ABTS (ELISA)
•Quantification - Used BioRad microplate spectrophotometer 3550-UV at 405nm with background substraction at 660nm
and analyzed with Microplate Manager v.2 software for Macintosh (BioRad)
•Ref: Dobak J, et al, 1,25-Dihydroxyvitamin D3 increases collagen production in dermal fibroblasts J Dermatol Sci 1994; 8:18
•Work done by – Sunny BioDiscovery, CA, USA
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Sytenol A Stimulates Collagens Type III
(Cell Culture Study; Old Fibroblasts)

Sytenol® A and Retinol: Increased collagen III output per cell without increasing cell numbers

Collagen III: % Stimulatory effects vs control

Quantification of collagenic protein: type III

•Cell Line – Normal human dermal fibroblasts; 68 yrs old; No cell proliferation seen with Sytenol® A & Retinol at 10 µg/ml
•Cell Culture - DMEM with 5% calf serum
•Use level – 10 µg/ml; dissolved in DMSO & then diluted in water; Water used as a control; Cells treated with the products for
7 days
•Protocol - Tested for type III collagen by sandwich ELISA using affinity-purified antibodies, followed by streptavidin-avidin-
HRP conjugate and ABTS (ELISA)
•Quantification - Used BioRad microplate spectrophotometer 3550-UV at 405nm with background substraction at 660nm and
analyzed with Microplate Manager v.2 software for Macintosh (BioRad)
•Ref: Dobak J, et al, 1,25-Dihydroxyvitamin D3 increases collagen production in dermal fibroblasts J Dermatol Sci 1994; 8:18
•Work done by – Sunny BioDiscovery, CA, USA

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Sytenol A has Inhibitory Activity Against
Pro-inflammatory Enzymes

 Weak inhibitor of phospholipase A2, but dose-dependently reduce formation of LTB4 and TXB2
• ML Ferandiz et.al., Effect of bakuchiol on leukocyte functions and some inflammatory
responses, J Pharm Pharmacol, 48(9), 975-980, 1996
• Effective inhibitor of edema and myeloperoxidase activity in the 12-O-
tetradecanoylphorbol 13-acetate (TPA)-induced ear edema
• Able to control leukocyte functions such as eicosanoid production, migration and
degranulation in the inflammatory site

 Good inhibitor of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activity

• IC50 of Sytenol A for COX-1 (14.7 µg/ml) and COX-2 (514 µg/ml)
• Measured by monitoring oxygen consumption using an Oxytherm electrode Unit from
Hansatech

• ~40% reduction in COX-2 activity using 50 µg/ml of Sytenol® A while Retinol boosts COX-2
activity
• Determined by using a kit from Cayman Chemical (cat. # 760151); Determined COX-2
activity at 590nm with BioRad 3550-UV microplate spectrophotometer

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Sytenol A has Excellent Elastase Inhibitory Activity

 What role Elastase play? % Reduction in Elastase activity

• Elastase breaks down elastin, an elastic fiber that,
together with collagen, determines the mechanical
properties of connective tissue
 Method
• Assayed using Calbiochem human neutrophil elastase
(Cat # 324681) & its substrate (Elastase substrate VIII).
• Positive control – El III (Calbiochem Catalog # 324745) &
negative control water
• Elastase inhibitory activity was determined by quantifying
the absorbance of chromophoric reaction product at 410
nm using BioRad microplate reader

DNA Microarray

Gene Description Fold Change

Retinol Sytenol A
Elastase-specific inhibitor (ESI) +2.5 +2.7

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Anti-Aging Clinical Study Protocol

• Human volunteers –17; 16 Completed; Caucasian (14), Hispanics (2); Age – 41 to 60

yrs
• Study duration – 12 weeks; May to August 2012 (winter in Australia)
• Test sites – Full face
• Test substances – Lotion with 0.5% Sytenol A; Contains No sunscreen and No
moisturizer
• Application frequency – About 2 g twice a day
• Methodology – Expert grading/Self-assessment by panelists (Grading 0 to 4): (1)
Roughness & Dryness; (2) Fine lines & wrinkles; (3) Skin tone; (4) Skin elasticity &
firmness; (5) Radiance; (6) Brightening; (7) Overall eye-area appearance; Silicone
Replica Analysis: (1) Wrinkle depth & (2) Skin roughness; Photography: Before & after
the treatments; Readings were taken at baseline, 4, 8 & 12 weeks.
• Results – Compared to initial day (pre-treatment)
• Statistical Analysis - Statistical significance defined as p=0.05 or less
• Work done by – Cantor Research Lab, Australia

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Study Results: % Improvement seen by Experts &
Panelists

% improvement by Expert % improvement by Panelists

p = <0.05
p = <0.05

1: Roughness & Dryness; 2: Fine lines & wrinkles; 3 Skin tone; 4: Skin elasticity & firmness

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Study Results: % Improvement seen by Experts &
Panelists

% improvement by Expert % improvement by Panelists

p = <0.05 p = <0.05

5: Radiance; 6:Brightening; 7: Eye area appearance

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Study Results: Silicone Replica Analysis to Quantify the
Reduction in Wrinkle Depth & Skin Roughness

% Reduction in Wrinkle depth & Skin roughness

-5

-10 4 wks
P = <0.05
8 wks
-15 12 wks

-20

-25

Protocol:
 At each visit, a single silicone replica was made of the target area and a photographic record was kept of this target for subsequent
relocation. The samples were stored in controlled conditions for comparative measurement. Comparative analysis of skin profilometry
was conducted using surface roughness and wrinkle depth analysis.
 The heights of the replicated wrinkles were measured using MiyomotoSurftestprofilometer. Ry (depth) and Ra (mean roughness)
were recorded at each time of measuring operation. The area scanned from each sample was clearly mapped so as to determine the
same area in respective of Week 4, Week 8, and Week 12 samples.

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Study Results: Photographic Comparison Between Before
& After Treatment

M572 Right View Pre Application M572 Right View 12 Weeks

Wrinkle Depth (µm)

BASE
4 WK 8 WK 12 WK

18.50 18.30 13.80 12.70

Skin Roughness (µm)

BASE 4 WK 8 WK 12 WK
3.10 4.05 2.91 2.42

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Study Results: Photographic Comparison Between Before
& After Treatment

M532 Right View Pre Application M532 Right View 12 Weeks

Wrinkle Depth (µm)

BASE
4 WK 8 WK 12 WK

38.60 30.40 29.80 23.90

Skin Roughness (µm)

BASE 4 WK 8 WK 12 WK
9.15 8.35 8.61 5.07

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Study Results: Photographic Comparison Between Before
& After Treatment: Reduction in wrinkles & hyper-pigmentation

M125 Left View Pre Application M125 Left View 8 Week

M125 Left View 4 Week M125 Left View 12 Week

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Study Results: Photographic Comparison Between Before
& After Treatment

M141 Left View Pre Application M141 Left View 8 Week

M141 Left View 4 Week M141 Left View 12 Week

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Study Results: Photographic Comparison Between Before
& After Treatment – Reduction in wrinkle & skin redness

M535 Frontal View Pre Application M535 Frontal View 8 Week

M535 Frontal View 4 Week M535 Frontal View 12 Week

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Sytenol A is a Very Effective Stabilizer for Retinol Under
Photo-oxidative Environment

 Complete photo-stabilization of Retinol using four-fold

excess of Sytenol A
 Provides option to use Retinol without worrying about
its instability; Added skin care benefits from Sytenol A

Protocol
 Retinol (50 µg/ml) was dissolved in ethanol and then Sytenol A
was added 50, 100 & 200 µg/ml using 5mL OPTICLEAR© vials
(KIMBLE glass, Chicago Heights, IL)
 Test vials were then placed in a Rayonet RPR-100 photochemical
reactor equipped with four RMR-3000 (UVB) and four RMR 3500
(UVA) lamps (Southern New England Ultraviolet Company, Branford,
CT), the combination of UVA and UVB simulated day light condition
 The samples were irradiated at 31 °C for 5 min at a dose of 13
J/cm2.
Retinol was quantified by HPLC using a 4.6 × 250 mm Luna C 18
column (Phenomenex, Torrance, CA) equipped with a DAD detector
at 280 nm
 The mobile phase consisted of 75% A (acetonitrile ) and 25% B
(methanol), isocratically ran at 1 ml/min for 10 minutes.

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Sytenol A is a Very Effective Stabilizer for Retinol Under
Singlet Oxygen Environment

 Complete stabilization of Retinol using only two-fold

excess of Sytenol A
 Provides option to use Retinol without worrying about
its instability; Added skin care benefits from Sytenol A

Protocol : Reference - Tetrahedron Letters 43 (2002) 8731–8734

• Retinol (50 µg/ml) was dissolved in ethanol and then
Sytenol A was added 50, 100 & 200 µg/ml using 5mL
OPTICLEAR© vials.
• Singlet oxygen was generated by addition of H2O2 and
Lithium molybdate in pH 9.01.
• The mixture was then incubated at 37˚C in dark for 15
hrs. During the period, singlet oxygen was generated and
retinol was oxidized.
• Retinol was quantified by HPLC on a 4.6 × 250 mm Luna
C 18 column (Phenomenex, Torrance, CA) using a DAD
detector with detection 280 nm.
• The mobile phase consisted of 75% A (acetonitrile ) and
25% B (methanol), isocratically ran at 1 ml/min.

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How to Formulate Products with Sytenol A?

 Sytenol A should be used at a level of 0.5 to 1.0% (w/w) of finished formulation; Easy
to develop creams, lotions, gels, sprays etc
 Sytenol is miscible in a wide variety of emolients, solubilizers, such as, Caprylic/capric
triglycerides, C 12-15 alkyl benzoates, ethyl linoleate, mineral oils, jojoba oils, olive oils,
dimethicones, cyclomethicones etc
 Add Sytenol A to the formulation after making emulsion with a processing
temperature of about 50˚C . Alternately, Sytenol® A can be included in the oil phase
 Contact with iron or copper compounds must be avoided as it is a phenolic compound,
which by nature tend to become colored in the presence of metal ions; Addition of a
small amount of chelating agent (~0.05%) resolves this problem.
 The finished product must be acidic, preferably having pH below 6.5
 The finished product must be protected from prolong exposure to heat and light

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Publications

1. RK Chaudhuri, The Miracle of Retinol; Why there is so much fuss about

retinoids and retinoid-like products? SPC, 23-24, 2010
2. RK Chaudhuri, K Bojanowski, F Marchio, Retinol and retinol-like
compounds in skin care, Expression Cosmetique, 227-233, 2010
3. RK Chaudhuri & F Marchio, Bakuchiol in the management of acne-
affected skin, C&T, 126(7):502-510, 2011
4. RK Chaudhuri & K Bojanowski, Bakuchiol: A Retinol-Like Functional
Compound Revealed by Gene Expression Profiling & Clinically Proven to
have Anti-Aging Effects, International J Cosmetic Science, 2014, in press
5. RK Chaudhuri, Bakuchiol: A Retinol-Like Functional Compound,
Modulating Multiple Retinol and Non-retinol Targets, In Cosmeceuticals
and Active Cosmetics, 3rd Edition, Eds. Raja K Sivamani, Jared Jagdeo,
Peter Elsner, Howard I Maibach, 2014 in press

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Availability of Sytenol A

 Available currently in 1 Kg size

 Sample size – 40 g

 For sample and literature, contact

• Sytheon Ltd, 315 Wootton Street, Boonton, NJ 07005
• Tel: 979-988-1076
• E-mail: info@sytheonltd.com
• Website: www.sytheonltd.com

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Summary

 While very effective in protecting skin, reversing aging and improving problem
skin; Retinol is problematic
 Serious side effects including skin irritation, skin sensitization and teratogenicity
 Photochemically and hydrolytically unstable
 Very difficult to formulate
 Sytenol A is a functionally equivalent compound manifesting comparable ant-
aging benefits without
 Safety issues
o Nonirritating, non-sensitizing and no known teratogenic or other health concerns
 Formulating difficulties
o Compatible with a wide variety of cosmetic ingredients for ease of formulating
 Instability problems
o Suitable for all-day use, including outdoors
 Sytenol A stabilizes Retinol very effectively both under oxidative- & photo-
oxidative environment
 Providing an option to use Retinol without worrying about its instability
 Added skin care benefits due to Sytenol A

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Thank You