Alicia Gimenez, Cristina Davi, Elena Canadas, Juan Cebrian, Raquel Delgado , Lipotec S.A.U.01.12.15
Due to its interface function between the body and the environment, the skin is constantly exposed to environmental damaging agents including sun rays, smoke and air pollutants. These elements can induce the generation of free radicals and other highly reactive chemical species that are very harmful to the cells and other components of the skin, and are involved in the development of extrinsic skin aging and in the acceleration of intrinsic aging.
Free radicals are chemical species capable of independent existence that contain one or more unpaired electrons. However, the term reactive species is wider and includes free radicals but also other related nonradical species. In general, reactive species have been long recognized as substantially harmful to biomolecules and reactive oxygen species (ROS) are the better-known of them, but reactive nitrogen species (RNS) and reactive carbonyl species (RCS) are becoming more and more characterized [1].
ROS include both oxygen free radicals and certain nonradical derivatives of oxygen that are oxidizing agents and/or are easily converted into radicals. Superoxide (O2•-) and hydroxyl (OH•) are examples of oxygen free radicals. Among non-radicals are hydrogen peroxide (H2O2), ozone (O3) and singlet oxygen (O21Δg). ROS, especially the hydroxyl radical, which is the most reactive neutral free radical, are capable of damaging biological macromolecules such as carbohydrates, proteins, lipids and DNA. The superoxide radical can give rise to other ROS and RNS. Singlet oxygen is an exceptionally reactive non-radical form of oxygen that can directly oxidize proteins, DNA and lipids. [1].
RNS include nitrogen-based free radicals, like nitric oxide (NO•), and non-radicals, like peroxynitrite (ONOO-). RNS can induce extensive cell damage by causing structural alteration of proteins and DNA, consequently inhibiting enzymatic activity and interfering with regulatory functions. Most RNS are derived from nitric oxide (NO•), which can form very reactive intermediates; i.e., the reaction between NO• and superoxide produces peroxynitrite [1]. Being one of the most powerful RNS, peroxynitrite reacts with a large number of molecules, including amino acids, nucleic acids, glucosaminoglycans, lipids and antioxidants. It can nitrate proteins by reacting with tyrosine residues and producing 3-nitrotyrosine. This irreversible modification of proteins compromises the interconversion between their phosphorylated and dephosphorylated states and hence can alter the activity of enzymes and receptors which are essential mechanisms of cellular regulation. Additionally, peroxynitrite can damage important constituents of the extracellular matrix (ECM), such as hyaluronic acid [2].
Multiple Sources of Oxidants: Internal and Environmental
Reactive species in the skin can originate from many different sources, which can be internal and external. As an internal source, cells generate free radicals as a part of their physiological processes. The aerobic metabolism that takes place in mitochondria is the major source of endogenous ROS [3].
Several environmental factors lead to the formation of oxidative chemicals, mainly sun radiation and air pollution. Ultraviolet (UV) radiation causes the generation of ROS that induce inflammation, breakdown of ECM components and DNA damage, consequently promoting photo-oxidative damage in the skin [4].
Air pollution is an alarming environmental problem and a public health concern that affects many regions in the world. It is a result of the introduction of harmful substances in the air. Air pollutants are mainly derived from combustion of fossil fuels for energy production, needed for transportation, industrial activity and so on. In addition, although with a smaller contribution to atmospheric contamination, cigarette smoke is a source of hazardous chemicals that can be generated very close to the skin. Major pollutants in the air include sulfur oxides, nitrogen oxides, carbon monoxide, ozone, heavy metals and organic aerosols. Nitrogen oxides can deposit into soils and water and their derived compounds include the RNS peroxynitrite. Organic aerosols can also generate free radicals. They contain powerful pollutants known as polycyclic aromatic hydrocarbons (PAH), such as pyrene and benzo[a]pyrene. Pyrene is one of the most abundant PAH in the atmosphere. When photoactivated by UVA or visible light, it induces the generation of ROS that can induce single strand DNA breaks [3].
Cosmetic Shield to Prevent Skin Aging
The antioxidant Lipochroman synthetic molecule (INCI name: Dimethylmethoxy chromanol) is structurally related to tocopherols. It has a dual scavenging capacity for both ROS and RNS, thus conferring a wide spectrum protection from reactive species.
Human normal keratinocytes were incubated with Dimethylmethoxy chromanol in the presence of pyrene. Next, the cells were irradiated with UVA/visible light (320-800 nm) for 69 seconds at 4°C. Finally, DNA breaks induced by photoactivated pyrene were analyzed by the alkaline comet assay, and the results were expressed as olive tail moment (OTM).
The pretreatment of the keratinocytes with the antioxidative ingredientpromoted a reduction in the levels of the DNA damage induced by light-activated pyrene, which could be observed as a reduction of the comet tails size (figure 1), indicative of reduced DNA breaks.
The protection provided by Dimethylmethoxy chromanol reached 99.3% (figure 2), significantly shielding skin cells from the genotoxic effects of air pollutants.
Prevention of Protein Inactivation by Nitration
RNS can nitrate the amino acid tyrosine in peptides and proteins in vivo, leading to the inactivation of proteins and receptors. The reaction of the RNS peroxynitrite with tyrosine gives 3-nitrotyrosine as the main product, which can be used as a biomarker for the presence of RNS.
The ability of Dimethylmethoxy chromanol to scavenge peroxynitrite was monitored by measuring the formation of 3-nitrotyrosine. Tyrosine and peroxynitrite were incubated alone (control) or with different concentrations of the active ingredient (0.02-0.12 mM). The formation of 3-nitrotyrosine was determined by high-performance liquid chromatography (HPLC).
The active ingredient inhibited the formation of 3-nitrotyrosine in a dose-dependent manner and up to -94.4% at the highest tested concentration (0.12 mM) (figure 3). Thus, it can potentially protect proteins from the loss of activity induced by nitration.
Long-Lasting Antioxidative Power
The antioxidative power method was used to assess the antioxidative power (AP) of Dimethylmethoxy chromanol. In this standardized method, the reducing activity of antioxidants against the radical diphenyl-picryl-hydrazyl (DPPH) is monitored at different times by electron spin resonance (ESR) spectroscopy until saturation, when all antioxidative molecules have reacted with the radical. The reaction time or reactivity (tr) and the AP parameters are typical values for each antioxidant. The AP is expressed in antioxidative units (AU), where 1 AU corresponds to the activity of 1 ppm solution of pure ascorbic acid (vitamin C).
Dimethylmethoxy chromanol or other recognized antioxidative agents were incubated with DPPH and the signal intensity decay was recorded during the reaction to obtain the AP parameters. Dimethylmethoxy chromanol showed a higher AP compared with other well-known antioxidants.
In addition, the AP and the reactivity of dimethylmethoxy chromanol after being stored in a solution were found to remain stable after 24 and 48 hours.
The antioxidative properties of dimethylmethoxy chromanol and of the commonly used antioxidant butylhydroxytoluene (BHT) were compared. Dimethylmethoxy chromanol showed an 82-fold higher AP and a 24-fold greater reactivity compared with BHT. Additionally, a cosmetic formulation containing 0.05% dimethylmethoxy chromanol and the formulation containing the same concentration of BHT were kept at two different temperatures, room temperature (RT) or 40ºC, for 1, 2 or 3 months. The formulation containing dimethylmethoxy chromanol showed better antioxidative properties throughout time with respect to the cream containing BHT. AP of the tocopherol analog was between 32- and 47-fold higher than that of BHT for a cosmetic formulation after 1 or 3 months respectively. When the formulations were kept at 40ºC, the AP of the cream with dimethylmethoxy chromanol was between 29- and 34-fold higher (1 and 3 months respectively) than in the formulation with BHT.
The skin is continuously exposed to sources of potentially harmful chemical species. When there is an imbalance between these reactive species and the physiological antioxidative defenses, oxidative stress occurs and it results in damage at different levels, causing the appearance of signs of aging. The topical application of antioxidants is an effective strategy to prevent this damage.
Lipochroman synthetic molecule is an antioxidant specifically designed to protect the skin from the effect of reactive species. Pretreatment of skin cells with the ingredient prevented DNA damage induced by the abundant air pollutant (pyrene). Furthermore, it proved to scavenge several different harmful species, including the RNS peroxynitrite. The antioxidative power of Lipochroman synthetic molecule was higher compared with other commonly used antioxidants, and it remained stable along time. Additionally, its antioxidative properties were better in cosmetic formulations.
In summary, this ROS and RNS scavenger ingredient protects skin cells from the irreversible damage caused by air pollutants and other sources of free radicals, preventing premature skin aging.
References:
1. Halliwell B. Reactive Species and Antioxidants. Redox Biology Is a Fundamental Theme of Aerobic Life. Plant Physiology, 141: 312–322, 2006.
2. Cebrián J., Messeguer A., Facino RM, Garcia Antón JM. New anti-RNS and -RCS products for cosmetic treatment. Int J Cosmet Sci, 27: 271-278, 2005.
3. Moldoveanu AM. Advanced Topics in Environmental Health and Air Pollution Case Studies. Chapter 6, Air Pollution, Reactive Oxygen Species (ROS), and Autonomic Nervous System Interactions Modulate Cardiac Oxidative Stress and Electrophysiological Changes. InTech; 2011.
4. Pillai S. Oresajo C, Hayward J. Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation - a review.Int J Cosm Sci, 27:17-34, 2005.
Free radicals are chemical species capable of independent existence that contain one or more unpaired electrons. However, the term reactive species is wider and includes free radicals but also other related nonradical species. In general, reactive species have been long recognized as substantially harmful to biomolecules and reactive oxygen species (ROS) are the better-known of them, but reactive nitrogen species (RNS) and reactive carbonyl species (RCS) are becoming more and more characterized [1].
ROS include both oxygen free radicals and certain nonradical derivatives of oxygen that are oxidizing agents and/or are easily converted into radicals. Superoxide (O2•-) and hydroxyl (OH•) are examples of oxygen free radicals. Among non-radicals are hydrogen peroxide (H2O2), ozone (O3) and singlet oxygen (O21Δg). ROS, especially the hydroxyl radical, which is the most reactive neutral free radical, are capable of damaging biological macromolecules such as carbohydrates, proteins, lipids and DNA. The superoxide radical can give rise to other ROS and RNS. Singlet oxygen is an exceptionally reactive non-radical form of oxygen that can directly oxidize proteins, DNA and lipids. [1].
RNS include nitrogen-based free radicals, like nitric oxide (NO•), and non-radicals, like peroxynitrite (ONOO-). RNS can induce extensive cell damage by causing structural alteration of proteins and DNA, consequently inhibiting enzymatic activity and interfering with regulatory functions. Most RNS are derived from nitric oxide (NO•), which can form very reactive intermediates; i.e., the reaction between NO• and superoxide produces peroxynitrite [1]. Being one of the most powerful RNS, peroxynitrite reacts with a large number of molecules, including amino acids, nucleic acids, glucosaminoglycans, lipids and antioxidants. It can nitrate proteins by reacting with tyrosine residues and producing 3-nitrotyrosine. This irreversible modification of proteins compromises the interconversion between their phosphorylated and dephosphorylated states and hence can alter the activity of enzymes and receptors which are essential mechanisms of cellular regulation. Additionally, peroxynitrite can damage important constituents of the extracellular matrix (ECM), such as hyaluronic acid [2].
Multiple Sources of Oxidants: Internal and Environmental
Reactive species in the skin can originate from many different sources, which can be internal and external. As an internal source, cells generate free radicals as a part of their physiological processes. The aerobic metabolism that takes place in mitochondria is the major source of endogenous ROS [3].
Several environmental factors lead to the formation of oxidative chemicals, mainly sun radiation and air pollution. Ultraviolet (UV) radiation causes the generation of ROS that induce inflammation, breakdown of ECM components and DNA damage, consequently promoting photo-oxidative damage in the skin [4].
Air pollution is an alarming environmental problem and a public health concern that affects many regions in the world. It is a result of the introduction of harmful substances in the air. Air pollutants are mainly derived from combustion of fossil fuels for energy production, needed for transportation, industrial activity and so on. In addition, although with a smaller contribution to atmospheric contamination, cigarette smoke is a source of hazardous chemicals that can be generated very close to the skin. Major pollutants in the air include sulfur oxides, nitrogen oxides, carbon monoxide, ozone, heavy metals and organic aerosols. Nitrogen oxides can deposit into soils and water and their derived compounds include the RNS peroxynitrite. Organic aerosols can also generate free radicals. They contain powerful pollutants known as polycyclic aromatic hydrocarbons (PAH), such as pyrene and benzo[a]pyrene. Pyrene is one of the most abundant PAH in the atmosphere. When photoactivated by UVA or visible light, it induces the generation of ROS that can induce single strand DNA breaks [3].
Cosmetic Shield to Prevent Skin Aging
The antioxidant Lipochroman synthetic molecule (INCI name: Dimethylmethoxy chromanol) is structurally related to tocopherols. It has a dual scavenging capacity for both ROS and RNS, thus conferring a wide spectrum protection from reactive species.
Human normal keratinocytes were incubated with Dimethylmethoxy chromanol in the presence of pyrene. Next, the cells were irradiated with UVA/visible light (320-800 nm) for 69 seconds at 4°C. Finally, DNA breaks induced by photoactivated pyrene were analyzed by the alkaline comet assay, and the results were expressed as olive tail moment (OTM).
The pretreatment of the keratinocytes with the antioxidative ingredientpromoted a reduction in the levels of the DNA damage induced by light-activated pyrene, which could be observed as a reduction of the comet tails size (figure 1), indicative of reduced DNA breaks.
The protection provided by Dimethylmethoxy chromanol reached 99.3% (figure 2), significantly shielding skin cells from the genotoxic effects of air pollutants.
Prevention of Protein Inactivation by Nitration
RNS can nitrate the amino acid tyrosine in peptides and proteins in vivo, leading to the inactivation of proteins and receptors. The reaction of the RNS peroxynitrite with tyrosine gives 3-nitrotyrosine as the main product, which can be used as a biomarker for the presence of RNS.
The ability of Dimethylmethoxy chromanol to scavenge peroxynitrite was monitored by measuring the formation of 3-nitrotyrosine. Tyrosine and peroxynitrite were incubated alone (control) or with different concentrations of the active ingredient (0.02-0.12 mM). The formation of 3-nitrotyrosine was determined by high-performance liquid chromatography (HPLC).
The active ingredient inhibited the formation of 3-nitrotyrosine in a dose-dependent manner and up to -94.4% at the highest tested concentration (0.12 mM) (figure 3). Thus, it can potentially protect proteins from the loss of activity induced by nitration.
Long-Lasting Antioxidative Power
The antioxidative power method was used to assess the antioxidative power (AP) of Dimethylmethoxy chromanol. In this standardized method, the reducing activity of antioxidants against the radical diphenyl-picryl-hydrazyl (DPPH) is monitored at different times by electron spin resonance (ESR) spectroscopy until saturation, when all antioxidative molecules have reacted with the radical. The reaction time or reactivity (tr) and the AP parameters are typical values for each antioxidant. The AP is expressed in antioxidative units (AU), where 1 AU corresponds to the activity of 1 ppm solution of pure ascorbic acid (vitamin C).
Dimethylmethoxy chromanol or other recognized antioxidative agents were incubated with DPPH and the signal intensity decay was recorded during the reaction to obtain the AP parameters. Dimethylmethoxy chromanol showed a higher AP compared with other well-known antioxidants.
In addition, the AP and the reactivity of dimethylmethoxy chromanol after being stored in a solution were found to remain stable after 24 and 48 hours.
The antioxidative properties of dimethylmethoxy chromanol and of the commonly used antioxidant butylhydroxytoluene (BHT) were compared. Dimethylmethoxy chromanol showed an 82-fold higher AP and a 24-fold greater reactivity compared with BHT. Additionally, a cosmetic formulation containing 0.05% dimethylmethoxy chromanol and the formulation containing the same concentration of BHT were kept at two different temperatures, room temperature (RT) or 40ºC, for 1, 2 or 3 months. The formulation containing dimethylmethoxy chromanol showed better antioxidative properties throughout time with respect to the cream containing BHT. AP of the tocopherol analog was between 32- and 47-fold higher than that of BHT for a cosmetic formulation after 1 or 3 months respectively. When the formulations were kept at 40ºC, the AP of the cream with dimethylmethoxy chromanol was between 29- and 34-fold higher (1 and 3 months respectively) than in the formulation with BHT.
The skin is continuously exposed to sources of potentially harmful chemical species. When there is an imbalance between these reactive species and the physiological antioxidative defenses, oxidative stress occurs and it results in damage at different levels, causing the appearance of signs of aging. The topical application of antioxidants is an effective strategy to prevent this damage.
Lipochroman synthetic molecule is an antioxidant specifically designed to protect the skin from the effect of reactive species. Pretreatment of skin cells with the ingredient prevented DNA damage induced by the abundant air pollutant (pyrene). Furthermore, it proved to scavenge several different harmful species, including the RNS peroxynitrite. The antioxidative power of Lipochroman synthetic molecule was higher compared with other commonly used antioxidants, and it remained stable along time. Additionally, its antioxidative properties were better in cosmetic formulations.
In summary, this ROS and RNS scavenger ingredient protects skin cells from the irreversible damage caused by air pollutants and other sources of free radicals, preventing premature skin aging.
References:
1. Halliwell B. Reactive Species and Antioxidants. Redox Biology Is a Fundamental Theme of Aerobic Life. Plant Physiology, 141: 312–322, 2006.
2. Cebrián J., Messeguer A., Facino RM, Garcia Antón JM. New anti-RNS and -RCS products for cosmetic treatment. Int J Cosmet Sci, 27: 271-278, 2005.
3. Moldoveanu AM. Advanced Topics in Environmental Health and Air Pollution Case Studies. Chapter 6, Air Pollution, Reactive Oxygen Species (ROS), and Autonomic Nervous System Interactions Modulate Cardiac Oxidative Stress and Electrophysiological Changes. InTech; 2011.
4. Pillai S. Oresajo C, Hayward J. Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation - a review.Int J Cosm Sci, 27:17-34, 2005.