Virtually everything that human cells need to maintain health requires energy. Each cell contains hundreds to thousands of mitochondria, and each mitochondrion contains multiple copies of mitochondrial DNA (mtDNA). Mitochondrial proteins take food molecules and combine them with oxygen to create chemical energy. This chemical energy produced by the mitochondria through the cellular respiration process is called adenosine triphosphate (ATP) (see Figure 1).
Generally the duration of life varies inversely with the rate of energy expenditure during life. Bio-gerontologist Denham Harman, PhD, the “father of the free radical theory of aging,” once suggested that mitochondria are the crucial component of cells whose rate of decline dictates the overall rate of aging.1 In addition to supplying cellular energy, mitochondria are involved in a range of other processes such as signaling, cellular differentiation and cell death, as well as controlling the cell’s cycle and growth.2
In human cells and eukaryotic cells in general, DNA is found in two cellular locations: inside the nucleus and inside the mitochondria. The generation of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide and hydroxyl radicals as by-products of mitochondrial oxidative phosphorylation (see Figure 2) damages mitochondrial macromolecules including the mtDNA, leading to deleterious mutations.
MtDNA damage is more extensive and persists longer than nuclear DNA damage in human cells,3 so when ROS have damaged the mitochondrial energy-generating apparatus beyond a functional threshold, rather than restoring it to a healthy condition, the mitochondria releases proteins that activate the caspase pathway, leading to aging and apoptosis.4 In skin, this aging is expressed in the form of wrinkles, loss of tone, thinning and abnormal collagen accumulation.
The human mitochondrial genome was sequenced in 1981, facilitating the isolation of mutant mtDNA and eventually its quantification. These experiments confirmed that mtDNA damage increases aging in humans and other mammals.5 Recent work by Trifunovic et al.6 also supports the mitochondrial theory of aging, showing that in prematurely aged mice, defective mitochondrial DNA polymerase is expressed. This work provides a causative link between mtDNA damage and aging phenotypes in mammals.6
Delivering Mitochondria Nutrition
An approach to the mitochondrial theory of aging, as will be described, is a technology focused on mitochondria-nourishing compounds. However, since the efficacy of functional ingredients fundamentally is determined by their delivery and influenced by the vehicle and molecules themselves,7 it is important first to consider the delivery aspect; in this case, the technology was encapsulated for controlled delivery.
A free-flowing powder of solid, hydrophobic nanospheres containing the mitochondria-nourishing compounds was further encapsulated in moisture-sensitive microspheres averaging 100–300 nm in diameter to deliver them to the epidermal level. These spheres adhere to skin as a result of lipophilic properties and electrostatic forces. While they are unable to penetrate the stratum corneum (SC), once they are deposited on skin, they form a reservoir, and moisture in the skin triggers the release of the nanospheres, which penetrate the lower layers of the SC. The incorporation of this controlled release system into anhydrous cosmetic formulations was found to provide moisture-triggered and prolonged release of the active ingredients (data not shown).
The nano-encapsulated, chirally activeingredientsa at the core of the described technology (see Chirality in Chemistry) were used to nourish mitochondria and stimulate the mitochondria support enzymes,8–10 to protect against endogenous and exogenous ROS, in turn slowing the aging of skin cells and other cellular material.
Spin-trap phenyl-butyl-nitrone (PBN): Research has shown that spin-trap PBN provides neuroprotective, cytoprotective, anti-inflammatory, oxidative stress recovery and free radical scavenging properties.11 Spin-traps originally were used to measure free radical activity both in vivo and in vitro by their ability to form stable complexes. Reactive free radicals are attracted and bound to the beta carbon atom in the spin trap, forming a spin adduct and effectively “trapping” the free radical, allowing the structure of the trapped radical to be deduced, and returning it to a normal orbit before it causes damage to the mitochondria.12 The material acts to prevent free radical damage caused endogenously through normal metabolic processes or exogenously by sources such as UV radiation, NO and other air pollutants by absorbing electrons as they spin out of control.
Coenzyme Q10 (CoQ10): CoQ10 or ubiquinone is a naturally occurring compound found in all cells in the human body that plays a key role in the mitochondria by converting food into energy. Ninety-five percent of all human body energy requirements are converted to ATP with the aid of CoQ10, and the depletion of CoQ10 levels in skin cells leads to the premature aging as well as a decrease in the formation of new cells.13, 14
Studies have shown that low doses of CoQ10 applied topically reduce oxidation and DNA double-strand breaks. In addition, CoQ10 supplements have been shown to extend the lifespan of human cells. In the described technology, CoQ10 stabilizes the CoQ10 content in the skin cells to increase their longevity.15
R-lipoic acid: Normally only the R-enantiomer of lipoic acid occurs naturally and in miniscule amounts in animal and plant tissues. Due to the difficulty and high cost of isolating natural R-alpha lipoic acid (ALA), studies and products were initially conducted and produced using synthetic lipoic acid. Unlike natural R-ALA, however, synthetic lipoic acid contains a 50/50 mixture of both R-ALA and S-ALA. Thus, most cosmetic products on the market containing ALA in fact contain both forms of lipoic acid—the synthetic S form and the natural R form.
However, Loffelhardt and co-workers have shown16 the S-enantiomer to have an inhibiting effect on the R-enantiomer since they are isomers, and with their atomic arrangements reversed, the biological activity of the R-enantiomer is substantially reduced, creating oxidative stress in human cells.
R-lipoic acid is water- and fat-soluble8 and therefore can neutralize free radicals both in membranes and within cells; it can also mimic other antioxidants and improve their performance by replenishing them.17 When an antioxidant neutralizes a free radical, it turns the radical into a stable form. In the chemical reaction that follows, the free radical is eventually passed off to lipoic acid or a glutathione molecule, which allows the original antioxidant to regenerate and continue to neutralize more free radicals while ALA washes out the offending free radical.18 This provides protection to the mitochondria.
In addition to its antioxidant function, R-lipoic acid has been shown to remodel collagen synthesis; to inhibit the abnormal attachment of sugar to protein and collagen, which makes skin inflexible and stiff (glycosylation); and to provide an anti-inflammatory function.19, 20 Research also has shown that R-lipoic acid increases the mitochondrial membrane potential of aged rats by up to 50%, compared with unsupplemented, aged rats.8
Adenine: Adenine is one of the two purine nucleobases used in forming nucleotides of the nucleic acids. In DNA, adenine binds to thymine via two hydrogen bonds to assist in stabilizing the nucleic acid structures. Some scientists have proposed that during the origin of life on Earth, the first molecule formed was adenine by polymerization reaction.21 Adenine performs a variety of roles in cellular biochemistry including cellular respiration in the form of energy-rich ATP and other cofactors such as nicotinamide adenine dinucleotideand flavin adenine dinucleotide, thus increasing the longevity and proper functioning of the mitochondria.22
Acetyl-L-carnitine (ALC): ALC regulates the mitochondrial cytochrome-C oxidase level in the human body, including skin cells; cytochrome-C oxidase is a vital component of cellular energy processes and is responsible for virtually all oxygen consumption in mammals.23 ALC transports long-chain acyl groups from fatty acids into the mitochondrial matrix so they can be broken down to acetate via β-oxidation to obtain usable energy through the citric acid cycle. Throughout human aging, carnitine concentration in cells diminishes, affecting energy production by the mitochondria. Recently reported data clarifies the role of ALC and the carnitine transport system in the interplay between peroxisomes and mitochondrial fatty acid oxidation.24
Thus, the combination of R-lipoic acid with ALC can significantly improve metabolic function; at the same time, this combination lowers oxidative stress and free radical production.25
The antioxidant potential and activity of the enzymes in the complexa were evaluated through the Oxygen Radical Absorbance Capacity (ORAC) assayb developed at the National Institute on Aging in the National Institutes of Health (NIH). This assay is based on a hydrogen atom transfer (HAT) reaction mechanism, which is relevant to human biology (see HAT Assays). The stimulation by antioxidant enzymes thus served as an indicator of the nutritional effects of the complex on the mitochondria.
The ORAC assay based on Glazer’s study measures the oxidative degradation of a fluorescent molecule (either β-phycoerythrin or fluorescein) after being mixed with free radical generators such as azo-initiator compounds. Azo-initiators are considered to produce peroxyl free radicals by heating, which damages the fluorescent molecule, resulting in the loss of fluorescence. Antioxidants protect the fluorescent molecule from oxidative degeneration, and this degree of protection is quantified using a fluorometer.
The water-soluble vitamin E analog Troloxc was used as a calibration standard and the ORAC result is expressed as micromole Trolox equivalent (TE)/g (see Figure 3).1 Caffeic acid also served as a calibration standard and the hydroxyl radical antioxidant capacity (HORAC) result is expressed as µmole caffeic acid equivalent (CAE)/g.2 The acceptable precision of the ORAC assay is 15% relative standard deviation.
Results of the ORAC assay indicated that the water-soluble or hydrophilic antioxidant capacity and the lipid-soluble or lipophilic antioxidant capacity of the text complex were 30 and 13 µmole TE/g, respectively. In addition, the HORAC and nitrogen radical absorbance capacity was found to be 7 µmole CAE/g and 0.3 µmole TE/g, respectively (see Figure 3).
The Singlet Oxygen Absorbance Capacity (SOAC) using α-tocopherol as a calibration standard showed that the tested complex possessed the antioxidant power of 198 µmole VtE/g—higher antioxidant power than anti-oxidants currently used in the cosmetics industry. As a comparison, apples, evaluated for their ORAC activity by the USDA, generally have measured 22.10 to 42.75 micromoles TE/g.
Stimulating SC Antioxidant Enzymes
The skin is constantly exposed to environmental sources, producing reactive oxygen species (ROS). To protect against oxidative damage, the skin is equipped with a large network of enzymatic antioxidant defense systems such as catalase, superoxide dismutase (SOD), and tissue GSH.26 In human SC, SOD and catalase are considered major antioxidant enzymes.27 In various skin disorders, the decreased antioxidant enzyme activity could be considered a marker for the increased susceptibility of the skin to external stimuli.28
The ability of the test complex to induce SC antioxidant enzymes was evaluated by the protocol standardized by Hellemans et al.27 In this study, 40 albino guinea pigs of the same age weighing 350–430 g were selected. The dorsal skin of the guinea pigs was washed and a 35 cm2 area was shaved before exposing it to a single dose of UVB (290–320 nm) irradiation. The total energy exposure of the guinea pig was 0.9 J/cm2. The irradiation time was approximately 30 sec.
The test complex was applied in the irradiated area and the antioxidant enzyme expression was studied by taking noninvasive tape strippings to determine SOD and catalase activity in guinea pig SC in vivo. In each study, 5 successive tape strippings were collected on the abdomen region, and stored at -80°C until analysis.
Detection of the catalase and SOD activity on the tape strippings from the SC was estimated as described by Giacomoni et al.29 and Hellemans et al.,27 respectively. The total protein amount on the tape stripping was quantified as the total amount of amino acids after acid hydrolysis at elevated temperature.
The GSH activity was estimated through Beutler et al.30 and showed an increase in SOD (see Figure 4a), catalase (see Figure 4b), and tissue GSH level (see Figure 4c) in skin cells, up to 3.33-, 4.3- and 2.7-fold higher than the control treatment.
Mitochondria are a key organelle to the health of skin, producing important proteins necessary to control the cell energy release process. Oxidative damage has been implicated as a major factor in the decline of physiological functions of the mitochondria, which leads to the aging process.
The free radical scavenging and antioxidant abilities shown in the data for the test complexa suggest its ability to scavenge a range of deleterious free radicals and stimulate antioxidant enzymes to delay the aging symptoms. In addition to its antioxidant mechanisms, the complex prompts DNA repair abilities and mitochondrial nutrition through mobilization of acyl groups from fatty acids, and enhanced ATP production. In short, these effects and formulation benefits suggest a new, mitochondrial approach for targeting young and mature skin alike. .
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