Biodynes™ 0 Urban Defense and Age Defense

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Biodynes™ 0 Urban Defense and Age Defense
PersonalCare
Biodynes™ 03 (patent pending)
Urban Defense and Age Defense
SAP Code#: 137180 CLS Code#: 520402-42
INCI Name: Saccharomyces Ferment Lysate Filtrate
Key Product Attributes:
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Enviromental Pollution Protection
Lipid Barrier Protection
Lipid Barrier Repair
DNA Protection
Skin Firming and Smoothing
Anti-Wrinkle
Ozone Protection Factor
Prevention of AGE Formation
Product Information
Ozone (O3) is a highly reactive form of molecular oxygen in which normal molecular oxygen combines with highly reactive oxygen radicals
to form transitory, but somewhat stable molecules containing three
oxygen atoms.1 Sources for ozone can be completely natural such as
the generation of ozone due to lightning strikes or formation of ozone
in the upper atmosphere through reaction of high energy solar plasma
with oxygen in the troposphere. However, ozone can also be generated
from more manmade sources including, in particular, urban smog,
and many cities actually monitor urban ozone levels as an indicator
of smog.2 Because ozone is a key component of human-produced
smog, it has been studied extensively for its respiratory health effects, particularly for people prone to asthma and people living in
urban populations. More recently, it has now been recognized that
ozone has detrimental effects on skin.3-11 In particular, it has been
established that ozone, while it can only penetrate a few microns into
the stratum corneum, will almost instantly react with vitamin C and
vitamin E reservoirs in the skin, rapidly depleting these important antioxidants. In addition, it has been shown that ozone will also rapidly
convert skin and cellular lipids into lipid peroxides that lead ultimately
to membrane and skin degradation. Studies have also demonstrated
that ozone can oxidize important proteins and nucleic acids.12
PersonalCare
Biodynes™ O3
Urban Defense and Age Defense
On the other hand, ozone is also known as a safer alternative to
chemicals in regards to disinfectant properties, sanitizing or purifying everything from medical equipment to food, water and even air!
Topical ozone skin treatments have reached popularity, touting to
promote exfoliation and skin whitening.
yellow circle represents an internal standard that is used in SDS PAGE
to indicate reproducibility of the electrophoresis). The gel on the left
was not stressed, the one on the right was stressed for fifteen minutes with ozone. The green circles show examples of proteins that
were present in the unstressed yeast (left) that visually faded as a
result of ozone stress, (right, a down regulation of these proteins).
The red circles in the ozone-stressed yeast lysate indicate proteins
that are upregulated as a result of the ozone stress compared to
the unstressed gel on the left. Many other less obvious examples
exist in these two gels, the examples above are only intended to
demonstrate that indeed ozone stress can influence the production
of proteins within a living system. More in-depth computer aided
analysis of these gels can help to identify the bulk of the changes
that have occurred.
For all of these reasons, ozone became a target for our Biodynes™
Yeast Platform.20 The theory being that if ozone was applied to the
growing microorganisms, a ‘stress’ would be created on the yeast
such that the yeast would need to respond with protective agents
to help it survive against the stress.21-24 It was based on this theory,
that Biodynes™ O3 was developed, using the influence of ozone to
create an anti-aging active for both hair and skin.
Manufacturing Process
Yeast Lysate Created by Ozone
In Situ and In Vitro Testing Summary
Biodynes™ O3 is manufactured using fermentation biotechnology in
which yeast are fed a nutrient media essential for development of
the yeast. During the cellular growth phase of the yeast, a sublethal
dose of ozone is applied to the yeast generated via a commercial
ozonator. The ozone is applied for a specific duration of time while
maintaining a viable cell count in the fermentation process. Upon
completion of the ozone application, the yeast cells are then lysed
to break open the cellular membrane and the key constituents of
the interior of the cell, including material from the cytoplasm and
the nuclear materials, are isolated.
Figure 2 outlines several studies which were conducted to evaluate
the benefits of Biodynes™ O3 both in situ and in vitro. A fundamental
in situ study confirmed that Biodynes™ O3 provides an oxidative protective mechanism against ozone preventing the degradation of a
brilliant blue, ozone sensitive dye, carmine. A 2% level of Biodynes™
O3 successfully protected the dye from fading to a light blue color,
during an ozone purge of the dye solution.
Testing on a full thickness tissue model showed that Biodynes™ O3
very effectively protects the lipid bilayer from cholesterol degradation following ozone exposure. The results from this study were so
compelling that Biodynes™ O3 appeared to actually promote cholesterol synthesis. A follow-up cell culture study confirmed that a 1%
Biodynes™ O3 treatment did promote cholesterol synthesis, actively
repairing or rebuilding the lipid bilayer.
2-D Electrophoresis of the Lysate
After isolation of the lysate, the changes that occurred in the protein
composition of the yeast were examined using 2-Dimensional Sodium
Dodecylsulfate-Polyacrylamide Gel Electrophoresis (2D SDS-PAGE). 25
The images of an unstressed (left) and an ozone stressed yeast lysate (right) are shown below (Figure 1). Careful examination of the
two gels side by side shows that the application of the ozone does
indeed cause up-regulation and down-regulation of certain proteins.
Unstressed Yeast Lysate
Fig.1
Additional testing on a full thickness tissue model generated data
substantiating that Biodynes™ O3 offers DNA protection by reducing
both 8-oxoguanine and TT Dimer formation following exposure of the
tissue model to ozone. At a 5% test level of Biodynes™ O3, complete
DNA protection was achieved in the 8-oxoguanine study.
Ozone Stressed Yeast Lysate
Study
Level of Biodynes™ O3
tested
Biodynes™ O3 Test Result
Carmine Dye Test
2%
Protected dye from degradation
Cholesterol Protection 16-19, 26
5%
Offered complete protection
and appeared to promote
cholesterol synthesis
Cholesterol Synthesis 27
1%
Promoted Cholesterol
synthesis
DNA Protection 8-oxoguanine
as marker 26
1% and 5%
1% reduced DNA damage, 5%
offered complete protection
Prevention of TT Dimer
1%
formation and 8-oxoguanine 26
Scans of Unstressed and Ozone Stressed Yeast Lysate
In Figure 1, visually obvious changes in protein composition as a
result of ozone stress are noted in the green and red circles (the
Fig.2
In Situ and In Vitro Testing Summary of Biodynes™ O3
2
Reduced levels of both,
more effective at reducing 8
oxoguanine
Environmental Ozone Protection Factor (OPF)
In this study, some well known antioxidants, vitamin C (sodium
ascorbate, NaAsc) and 70% EGCG Green Tea Polyphenols (EGCG),
were compared against Biodynes™ O3 to determine the ability of the
products to prevent the formation of AGEs. As shown in Graph 2,
all the antioxidants provide some protection against AGE formation,
but Biodynes™ O3 was superior when compared to equal quantities
of 70% EGCG and about equal to vitamin C as a protectant.
Percentage of Protection
The data generated from the DNA Protection Study provides us with
an opportunity to develop a new definition of skin protection called
the “Ozone Protection Factor” or OPF. The results of this study can
be directly correlated to the ability of Biodynes™ O3 to protect skin
from environmental ozone exposure. The control cells that were not
treated with Biodynes™ O3 represent an OPF of 0%; as a significant
increase in 8-oxoguanine results from direct ozone exposure.  Incorporation of 1% Biodynes™ O3 offers the cells a moderate OPF in the
range of 50%; as the presence of Biodynes™ O3 effectively reduces
the level of 8-oxoguanine compared to the control. Likewise, 5% treatment of Biodynes™ O3 offers a high OPF in the range of 100%. These
values are plotted below as the anticipated performance of various
concentrations of Biodynes™ O3 to protect the skin from a day of
ozone exposure, Graph 1.
100
90
High
80
Medium
70
60
Graph 2
Anticipated Ozone Protection Factor For Various Concentrations of Biodynes™ O3
50
40
Low
In Vivo Panel Study
30
In order to evaluate the impact of Biodynes™ O3 on human skin, a
comprehensive in vivo study was initiated in São Paulo, Brazil, a city
that is known to have high levels of ozone during the summer time.28
A 60-person, double-blind, placebo controlled study was conducted
for a 90-day evaluation. The study was monitored by a skilled dermatologist who examined the participants throughout the course
of the evaluation.
20
10
0
1
2
3
4
5
Percentage Biodynes™ O3
Graph 1
Anticipated Ozone Protection Factor For Various Concentrations of Biodynes™ O3
In Vitro AGE Prevention – Full Thickness Tissue Model
Advanced Glycation Endproducts, or AGEs, form when oxidatively
damaged proteins such as collagen, elastin, keratin, fibronectin, etc.
react with sugars present in the skin to initially form some intermediate products called Shiff Bases and Amadori products, but ultimately
form irreversible products called Advanced Glycation Endproducts.29
It is generally felt that accumulation of AGEs in the skin is a measure
of protein breakdown due to oxidative stress and levels of AGEs have
been shown to be high in people with photoaged skin versus people
with less photoaged skin. Also, it has been demonstrated that aged
cells such as fibroblasts will have higher levels of AGEs than young
cells. Therefore, the formation of AGEs is an indication of skin aging
and extracellular matrix breakdown, which can ultimately lead to
sagging and wrinkles.
The participants were broken down into three groups of twenty. One
group was the placebo control group who used an SPF 8 lotion that
contained none of the test material. The second group used the
same lotion with 3% Biodynes™ O3 added and the third group applied
a 5% Biodynes™ O3 lotion. Each participant had a three day wash out
period prior to starting the study in which they were asked to stop
using their standard moisturizers and were asked to wash their faces
and arms with a non-conditioning bar soap. The participants were
not allowed to use any skin-effecting pharmaceuticals during the
course of the study.
On Day 90, at completion of the test period, the participants were
tested in the following ways: Facial moisturization using corneometer, forearm barrier integrity using TEWL, wrinkles using SilFlo®
replicants and digital analysis and dermatologist assessment of
skin firmness, overall facial elasticity and skin appearance.
An in vitro study was done on full thickness tissue (Epiderm EFT 200)
to evaluate the effects of ozone on influencing AGE production. As
demonstrated in Figure 3, compared to non-ozonated tissue (UTOzone), tissue treated with a short exposure to ozone shows a very
significant increase in AGE production (UT + Ozone).
3
PersonalCare
Biodynes™ O3
Urban Defense and Age Defense
Forearm Barrier Integrity
Ozone levels in São Paulo, Brazil from July to November 2005 were
monitored weekly. The data is shown in Graph 3.
Forearm barrier integrity was measured using transepidermal water
loss. Graph 5 shows the data for this study after the 90-day treatment.
Ozone Concentration (µg/m3)
Ozone Levels Throughout the 90 Day Study
140.000
120.000
100.000
80.000
60.000
40.000
20.000
0.000
1
2
3
4
5
6
7
8
9
10
Average 03
11
12
13
14
Fig.6
15
Time / Week
Graph 3
Weekly ozone levels in São Paulo, Brazil from July through November, 2005, as monitored by
CETESB (Companhia de Tecnologia de Saneamento Ambiental).
Graph 5
Effects on forearm TEWL after 90 days of treatment.
Results of In Vivo Testing
Facial Moisturization
From the data, it can be seen that after 90 days, the placebo-treated
control group showed a 92% decrease in barrier integrity compared
to 20% improvement in barrier integrity for the participants using
the lotion containing 5% Biodynes™ O3.
Changes in skin moisturization were determined using corneometric
analysis. Results are shown in Graph 4.
Wrinkle Study
At the end of the 90-day treatment period, participants were analyzed for wrinkle effects using Silflo replicants and digital photography to assess overall wrinkle appearance. The data indicates that
34% of the participants using 5% Biodynes™ O3 showed a statistically
significant improvement in overall wrinkles compared to the placebotreated controls. A 5% treatment level showed the best performance
in cutaneous relief, improvement in skin roughness, and improvement in deep wrinkles.
Dermatological Assessment Facial Skin Firmness
After the 90 day treatment period, the participants were examined
by a dermatologist who assessed their skin for facial firmness. The
data from this examination is provided in Graph 6.
Graph 4
Improvements in facial moisturization after 90 days of treatment.
Dermatological assessment indicated a statistically significant improvement in skin firmness after 90 days, with a 72% improvement
in the group using 5% Biodynes™ O3 versus a 38% improvement for
the placebo-treated control group.
From the data it can be seen that after the 90-day treatment regimen,
the Biodynes™ O3 at 3% provided an 11% increase in moisturization
compared to an 8% improvement noted for the placebo-treated group.
4
Improvement in Skin Elasticity
80
% Improvement
% Improvement
% Improvement in Skin Firmness
70
60
80
70
60
50
50
40
40
30
30
20
20
10
10
0
30
60
0
90
30
60
Time in Days
placebo
3% Biodynes™ O3
5% Biodynes™ O3
placebo
Graph 6
Dermatological assessment of facial skin firmness
All data is statistically significant (p< 0.00001)
3% Biodynes™ O3
Graph 8
Dermatological assessment of skin elasticity. All data is statistically significant (p< 0.00001)
Dermatological assessment after 90 days indicated that at 3%
Biodynes™ O3, there was a statistically significant 73% increase in
skin elasticity versus 20% for the placebo controls.
AGE Study
Each participant in the study was tape stripped at baseline and at the
end of the 90 day treatment period, and tape strips were analyzed
for the presences of AGEs (Advanced Glycation Endproducts). The
data in Graph 7 indicates a dose response for reduction in AGEs going from the placebo formulation [No Active] to 3% and then to a 5%
Biodynes™ O3 treatment. The linear reduction in AGE formation in the
skin over the 90-day test period confirms what was found in vitro on
the full thickness tissue model.
Dermatological Assessment of Overall Skin Appearance
After 90 days, the participants were assessed by a dermatologist for
their overall facial skin appearance. Results are shown in Graph 9.
% Improvement
% Improvement in Overall Skin Appearance
Change in AGE Content
0
Change in AGE Fluorescence (Day 0 - Day 90)
90
Time in Days
80
70
60
50
-1
40
-2
30
20
-3
10
0
-4
30
60
90
Time in Days
placebo
-5
Treatment Group
placebo
3% Biodynes™ O3
3% Biodynes™ O3
Graph 9
Dermatological assessment of overall skin appearance.
All data is statistically significant (p< 0.00001)
5% Biodynes™ O3
Graph 7
Dermatological Assessment of Skin Elasticity
Dermatological assessment after 90 days indicated that at a level of
3% Biodynes™ O3 there was a statistically significant 76% increase
in overall skin firmness versus 30% for the placebo controls.
Following the 90-day treatment, the participants were assessed by
a dermatologist for changes in their facial skin elasticity. The results
of this assessment are shown in Graph 8.
5
PersonalCare
Biodynes™ O3
Urban Defense and Age Defense
Typical Properties
Day 0: Panelist 1: 5% Biodynes™ O3
Day 90: Panelist 1: 5% Biodynes™ O3
Appearance
Clear, yellowish liquid
pH (Direct @25° C)
4.0 – 7.0
Non-Volatile Matter (1g - 1hr - 105° C)
1.0 – 4.0%
Residue on Ignition
1.5% Maximum
Nitrogen (Kjeldahl)
0.1 – 1.0%
Microbial Content
100 opg Maximum; No pathogens
Preservative System
0.9 – 1.1% Phenoxyethanol
Recommended Use Level
0.5 - 3.0%
Product Safety Review
Day 0: Panelist 2: 5% Biodynes™ O3
Day 90: Panelist 2: 5% Biodynes™ O3
Fig #
Conclusion
Biodynes™ O3 is a yeast extract that offers one of the first, scientifically-designed, topically applied defenses against ozone, which
is rapidly becoming a well recognized threat against skin health,
aging and well being. In vitro studies confirm that Biodynes™ O3 is
effective at protecting fragile cellular nucleic acids, including nuclear
and mitochondrial DNA and RNA, and skin lipids, critical for proper
functioning of the skin’s lipid structure, from the detrimental effects
of ozone. In vitro studies also confirm Biodynes™ O3 prevents the formation of Advanced Glycation Endproducts as effectively as green
tea and vitamin C.
In vivo clinical studies substantiate the use of Biodynes™ O3 as an
active affording anti-wrinkle, skin firming claims as well as reducing
trans-epidermal water loss and enhancing skin moisturization, and
reducing AGE formation.
Biodynes™ O3 allows a formulator to literally “dial in” a factor of ozone
protection (OPF) into their product depending on anticipated exposure times that the consumer may need the protection. Biodynes™ O3
can help assure the consumer that they are receiving steps towards
proper care and safeguards for the skin from harmful effects of the
environment; it truly is the pollution solution!
4
Epi-Ocular – MTT Viability Assay
(Product tested at 100%)
Minimal/to mild
Epi-Derm – MTT Viability Assay
(Product tested at 100%)
Minimal/to mild
RIPT
Non-sensitizing
References
16.Smith LL. Oxygen, Oxysterols, Ouabain and Ozone: A Cautionary
Tale. Free Rad Biol Med 37; 318-324, 2004.
17.Girotti AW. Photosensitized Oxidation of Cholesterol in Biological
Systems and Reaction Pathways, Cytotoxic Effects and Defense
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Detector in Biological Systems. Method Enzymol 319; 85-100,
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19.Jans R, Atanasova G, Jadot M, Poumay Y. Cholesterol
Depletion Upregulates Involucrin Expression in Epidermal
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20.Venkatesan VP, Gruber JV. Biodynes TRF for Personal Care and
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27, 2006), EBf880a (Dec. 20, 2005), EBf880c (Mar. 13, 2006)
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to Ozone Depletes Vitamins C and E and Induces Lipid Peroxidation
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5
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This product information corresponds to our knowledge on the subject at the date of publication and we
assume no obligation to update it. It is offered without warranty, and is intended for use by persons who
are experienced and knowledgeable in the field and capable of determining the suitability of ingredients for
their specific applications. Because we cannot anticipate all variations in actual end-use conditions, we assume no liability and make no warranty in connection with your use of our products or product information.
We do not guarantee the efficacy of active ingredients, delivery systems, functional ingredients, rheology
modifiers, natural or botanical products, preservative and protection systems or proteins in any specific
application or use. The information we provide is not intended to substitute for testing. You should perform
your own tests to determine for yourself the suitability and efficacy of ingredients in your application and
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