PersonalCare 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 ef- fects, 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 an- tioxidants. 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 INCI Name: Saccharomyces Ferment Lysate Filtrate SAP Code#: 137180 CLS Code#: 520402-42 Key Product Attributes: – 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 (O 3 ) is a highly reactive form of molecular oxygen in which nor- mal 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 Biodynes™ 0 3 (patent pending) Urban Defense and Age Defense
Transcript
PersonalCare
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 ef-fects, 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 an-tioxidants. 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
Key Product Attributes: – 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 InformationOzone (O
3) is a highly reactive form of molecular oxygen in which nor-
mal 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
Biodynes™ 03 (patent pending)
Urban Defense and Age Defense
PersonalCareBiodynes™ O
3
Urban Defense and Age Defense
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On the other hand, ozone is also known as a safer alternative to chemicals in regards to disinfectant properties, sanitizing or purify-ing everything from medical equipment to food, water and even air! Topical ozone skin treatments have reached popularity, touting to promote exfoliation and skin whitening.
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™ O
3 was developed, using the influence of ozone to
create an anti-aging active for both hair and skin.
Manufacturing ProcessYeast Lysate Created by OzoneBiodynes™ O
3 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.
2-D Electrophoresis of the LysateAfter 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 ly-sate (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.
Fig.1
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
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 min-utes 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.
In Situ and In Vitro Testing SummaryFigure 2 outlines several studies which were conducted to evaluate the benefits of Biodynes™ O
3 both in situ and in vitro. A fundamental
in situ study confirmed that Biodynes™ O3 provides an oxidative pro-
tective mechanism against ozone preventing the degradation of a brilliant blue, ozone sensitive dye, carmine. A 2% level of Biodynes™ O
3 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 degrada-tion following ozone exposure. The results from this study were so compelling that Biodynes™ O
3 appeared to actually promote choles-
terol synthesis. A follow-up cell culture study confirmed that a 1% Biodynes™ O
3 treatment did promote cholesterol synthesis, actively
repairing or rebuilding the lipid bilayer.
Additional testing on a full thickness tissue model generated data substantiating that Biodynes™ O
3 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™ O
3, complete
DNA protection was achieved in the 8-oxoguanine study.
Fig.2In Situ and In Vitro Testing Summary of Biodynes™ O
1% and 5% 1% reduced DNA damage, 5% offered complete protection
Prevention of TT Dimer formation and 8-oxoguanine 26
1% Reduced levels of both, more effective at reducing 8 oxoguanine
3
Environmental Ozone Protection Factor (OPF)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™ O
3 to protect skin
from environmental ozone exposure. The control cells that were not treated with Biodynes™ O
3 represent an OPF of 0%; as a significant
increase in 8-oxoguanine results from direct ozone exposure. Incor-poration of 1% Biodynes™ O
3 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% treat-ment of Biodynes™ O
3 offers a high OPF in the range of 100%. These
values are plotted below as the anticipated performance of various concentrations of Biodynes™ O
3 to protect the skin from a day of
ozone exposure, Graph 1.
Graph 1Anticipated Ozone Protection Factor For Various Concentrations of Biodynes™ O
3
In Vitro AGE Prevention – Full Thickness Tissue ModelAdvanced 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 intermedi-ate 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.
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 (UT-Ozone), tissue treated with a short exposure to ozone shows a very significant increase in AGE production (UT + Ozone). In this study, some well known antioxidants, vitamin C (sodium ascorbate, NaAsc) and 70% EGCG Green Tea Polyphenols (EGCG), were compared against Biodynes™ O
3 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™ O
3 was superior when compared to equal quantities
of 70% EGCG and about equal to vitamin C as a protectant.
Graph 2
In Vivo Panel StudyIn order to evaluate the impact of Biodynes™ O
3 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 der-matologist who examined the participants throughout the course of the evaluation.
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™ O
3 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.
Percentage of Biodynes™ O3
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Change in Autofluorescence with Ozone Exposure (AGE Measurement)
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Urban Defense and Age Defense
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On Day 90, at completion of the test period, the participants were tested in the following ways: Facial moisturization using corneom-eter, forearm barrier integrity using TEWL, wrinkles using SilFlo® replicants and digital analysis and dermatologist assessment of skin firmness, overall facial elasticity and skin appearance.
Ozone Levels Throughout the 90 Day StudyOzone levels in São Paulo, Brazil from July to November 2005 were monitored weekly. The data is shown in Graph 3.
Graph 3Weekly ozone levels in São Paulo, Brazil from July through November, 2005, as monitored by CETESB (Companhia de Tecnologia de Saneamento Ambiental).
Results of In Vivo TestingFacial MoisturizationChanges in skin moisturization were determined using corneometric analysis. Results are shown in Graph 4.
Graph 4Improvements in facial moisturization after 90 days of treatment.
From the data it can be seen that after the 90-day treatment regimen, the Biodynes™ O
3 at 3% provided an 11% increase in moisturization
compared to an 8% improvement noted for the placebo-treated group.
Forearm Barrier IntegrityForearm barrier integrity was measured using transepidermal water loss. Graph 5 shows the data for this study after the 90-day treatment.
Graph 5Effects on forearm TEWL after 90 days of treatment.
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™ O
3.
Wrinkle StudyAt the end of the 90-day treatment period, participants were ana-lyzed for wrinkle effects using Silflo replicants and digital photog-raphy to assess overall wrinkle appearance. The data indicates that 34% of the participants using 5% Biodynes™ O
3 showed a statistically
significant improvement in overall wrinkles compared to the placebo-treated controls. A 5% treatment level showed the best performance in cutaneous relief, improvement in skin roughness, and improve-ment in deep wrinkles.
Dermatological Assessment Facial Skin FirmnessAfter 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.
Dermatological assessment indicated a statistically significant im-provement in skin firmness after 90 days, with a 72% improvement in the group using 5% Biodynes™ O
3 versus a 38% improvement for
the placebo-treated control group.
AverageTime/Week
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Graph 6Dermatological assessment of facial skin firmnessAll data is statistically significant (p< 0.00001)
AGE StudyEach 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 go-ing from the placebo formulation [No Active] to 3% and then to a 5% Biodynes™ O
3 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.
Graph 7
Dermatological Assessment of Skin ElasticityFollowing 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.
Graph 8Dermatological assessment of skin elasticity. All data is statistically significant (p< 0.00001)
Dermatological assessment after 90 days indicated that at 3% Biodynes™ O
3, there was a statistically significant 73% increase in
skin elasticity versus 20% for the placebo controls.
Dermatological Assessment of Overall Skin AppearanceAfter 90 days, the participants were assessed by a dermatologist for their overall facial skin appearance. Results are shown in Graph 9.
Graph 9 Dermatological assessment of overall skin appearance. All data is statistically significant (p< 0.00001)
3% Biodynes™ O35% Biodynes™ O
3
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Placebo3% Biodynes™ O3
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Urban Defense and Age Defense
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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.
Fig #
ConclusionBiodynes™ O
3 is a yeast extract that offers one of the first, scien-
tifically-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™ O
3 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™ O
3 prevents the for-
mation 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 expo-sure times that the consumer may need the protection. Biodynes™ O
3
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!
Day 0: Panelist 1: 5% Biodynes™ O3
Day 90: Panelist 1: 5% Biodynes™ O3
Day 0: Panelist 2: 5% Biodynes™ O3 Day 90: Panelist 2: 5% Biodynes™ O
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
5
References1. Gaffner JS, Marley NA, Atmospheric Chemistry and Air Pollution. Sci World J 3; 199-234, 2003.2. US Environmental Protection Agency website: http://www.epa.gov/airtrends/ozone.html.3. Thiele JJ, Traber MG, Polefka TG, Cross CE, Packer L. Ozone- Exposure Depletes Vitamin E and Induces Lipid Peroxidation in Murine Stratum Corneum. J Invest Dermatol 108; 753-757, 1997.4. Thiele JJ, Traber MG, Tsang K, Cross CE, Packer L. In Vivo Exposure to Ozone Depletes Vitamins C and E and Induces Lipid Peroxidation in Epidermal Layers of Murine Skin. Free Rad Biol Med 23; 385-391, 1997.5. Weber SU, Jothi WS, Thiele JJ. High Pressure Liquid Chromato graphic Analysis of Ozone-Induced Depletion of Hydrophilic and Lipophilic Antioxidants in Murine Skin. Method Enzymol 319; 536- 546, 2000.6. Weber SU, Han N, Packer L. Ozone: An Emerging Oxidative Stressor to Skin. Curr Probl Dermatol 29; 52-61, 2001.7. Cotovio J, Onno L, Justine P, Lamure S, Catroux P. Generation of Oxidative Stress in Human Cutaneous Models Following In Vitro Ozone Exposure. Toxicol in Vitro 15; 357-362, 2001. 8. Packer L, Valacchi G. Antioxidants and the Response of Skin to Oxidative Stress: Vitamin E as a Key Indicator. Skin Pharmacol Appl Skin Physiol 15; 282-290, 2002. 9. Thiele JJ, Dreher F, Maibach HI, Packer L. Impact of Ultraviolet Radiation and Ozone on the Transepidermal Water Loss as a Function of Skin Temperature in Hairless Mice. Skin Pharmacol Appl Skin Physiol 16; 283-290, 2003. 10. Valacchi G, Pagnin E, Corbacho AM, Olano E, Davis PA, Packer L. In Vivo Ozone Exposure Induces Antioxidant/Stress Related Responses in Murine Lung and Skin. Free Radic Biol Med 36; 673- 681, 2004.11. Tavakkol A, Nabi Z, Cardona S, Soliman N, Polefka T. Tropospheric Ozone Induces Lipid Hydroperoxides and Depletes Vitamin E in Human Skin In Vivo with no Detectable Clinical or Biophysical Changes. J Invest Dermatol 114; 844, 2000.12. Victorin K. Review of the Genotoxicity of Ozone. Mutat Res 277; 221-228, 1992.13. Krutmann J, Premature Skin Aging by Ultraviolet Radiation and Other Environmental Hazards: The Molecular Basis. Hautarzt 54; 809-817, 2003.14. He QC, Krone K, Schert D, Kotler M, Tavakkol A. The Use of Ozone as an Oxidizing Agent to Evaluate Antioxidant Activitites of Natural Substrates. Skin Pharmacol Physiol 17; 183-189, 2004.15. Wentworth P, Nieva J, Takeuchi C, Gaive R, Wentworth AD, Diley RB, DeLaria GA, Saven A, Babior BM, Janda KD, Eschenmoser A, Lerner RA. Evidence for Ozone Formation in Human Atherosclerotic Arteries. Science 302; 1053-1056, 2003.
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 Mechanisms. J Photochem Photobiol B: Biol 13; 105-118, 1992.18. Girotti AW, Korytowski W. Cholesterol as a Singlet Oxygen Detector in Biological Systems. Method Enzymol 319; 85-100, 2000.19. Jans R, Atanasova G, Jadot M, Poumay Y. Cholesterol Depletion Upregulates Involucrin Expression in Epidermal Keratinocytes Through Activation of p38. J Invest Dermatol 123; 564-573, 2004.20. Venkatesan VP, Gruber JV. Biodynes TRF for Personal Care and Cosmetics. Spec Chem Mag 24; 19; 2004.21. Dubeau H, Chung YS. Ozone Response in Wild Type and Radiation- Sensitive Mutants of Saccharomyces cerevisiae. Molec Gen Genet 176; 393-398, 1979.22. Hinze H, Prakash D, Holzer H. Effect of Ozone on ATP, Cytosolic Enzymes and Permaeability of Saccharomyces cerevisiae. Arch Microbiol 147; 105-108, 1987.23. Cabiscol E, Piulats E, Echave P, Herrero E, Ros E. Oxidative Stress Promotes Specific Protein Damage in Saccharomyces cerevisiae. J Biol Chem 275; 27393-27398, 2000.24. Thorpe GW, Fong CS, Alic N, Higgins VJ, Dawes IW. Cells Have Distinct Mechanisms to Maintain Protection Against Different Reactive Oxygen Speices: Oxidative-Stress Response Genes. PNAS 101; 6564-6569, 200425. Maillet I, Lagniel G, Perrot M, Boucherie H, Labarre J. Rapid Indentification of Yeast Proteins on Two-Dimensional Gels. J Biol Chem 271; 10263-10270, 1996.26. Arch Personal Care Products, Ozone Damage Protection Study, Study Numbers 04-0041 and 04-0059, Sept. 4, 2004.27. Arch Personal Care Products, EpiDerm Cholesterol Synthesis Assay, Study Number 04-0074, Feb. 15, 2005.28. Arch Personal Care Products, In vivo study conducted by EVIC Brasil, Study Numbers EBf880b (Dec. 20, 2005), EBf880d (Jan. 27, 2006), EBf880a (Dec. 20, 2005), EBf880c (Mar. 13, 2006)29. Bucala R et al., Advanced Glycosylation: Chemistry, Biology, and Implications for Diabetics And Aging. Adv in Pharm 23 (1992) 1-34.
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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 as-sume 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 conditions of use. The information we provide should not be construed as a license to operate under or a recommendation to infringe any patent or other intellectual property right, and you should ensure that your use does not infringe any such rights. Our products are for industrial use only. WE MAKE NO WARRANTY (INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR PURPOSE) OF ANY KIND, EXPRESS OR IMPLIED, OTHER THAN THAT OUR PRODUCTS CONFORM TO THE APPLICABLE PRODUCT SPECIFICATIONS.
