Skin is constantly under attack by various oxidative stresses; external such as UV radiation, and internal such as reactive oxygen species (ROS) molecules generated during cell metabolism. Consequently, oxidative stress is recognized as a key factor in skin aging and constitutes a major concern in the cosmetic field. Since coenzyme Q10 (CoQ10) is a known powerful anti-oxidant that can play an essential role in mitochondrial energy synthesis,1 a new anti-oxidant, referred to here as compound IV08.004, based on pentapeptide-34 trifluoroacetatea was designed to activate CoQ10. Here, the authors describe both in vitro and in vivo studies investigating the compound’s anti-oxidant and anti-aging effects.
To increase CoQ10, poly PrenylTransferase (PDSS1/2) and CoQ10B were targeted; to understand the roles of these entities in CoQ10 production and CoQ10 itself in anti-aging, some background would first be helpful. In cells, CoQ10 is synthetized in the mitochondria starting from acetyl-CoA and tyrosine in a 17-step process (see Figure 1). The benzoquinone portion of CoQ10 is synthesized from tyrosine, whereas the isoprene side chain arises from acetyl-CoA through the mevalonate pathway. The enzyme Prenyl Diphosphate Synthase, constituted by PDSS1 and PDSS2 subunits—also known as Decaprenyl Diphosphate Synthase or Trans-Prenyl Transferase (TPT)—catalyzes the second step in the final reaction sequence of CoQ10 biosynthesis, which is the condensation of the poly-isoprenoid side chain with para-hydroxybenzoate.2
In relation, researchers recently identified a molecule closely related in structure to CoQ10: the Coenzyme Q-binding protein or CoQ10 homolog B.3 Functionally, based on similarity of the sequence, it should interact with CoQ10. This binding protein is required for the function of CoQ10 in the respiratory chain, and may serve as a chaperone or be involved in the transport from its site of synthesis to the catalytic sites of the respiratory complexes. Its presence is strongly correlated with the presence of CoQ10 itself.
In cells, the Mitochondrial Respiratory Chain (MRC), consisting of four multi-subunit complexes, collects metabolites mainly from the Krebs cycle, from pyruvate oxidation, amino acid and fatty acid catabolism to finally synthesize the high-energy compound adenosine-triphosphate (ATP). CoQ10, also known as ubiquinone, plays an essential role in the MRC as an electron carrier. Indeed, CoQ10 is the cofactor for the mitochondrial respiratory complexes I, II and III, functioning as an electron carrier from enzyme complex I and II, to complex III. These mitochondrial enzymatic complexes, being part of the oxidative phosphorylation pathway, are essential for the production of ATP, upon which all cellular functions depend.4
CoQ10 is present in all cell membranes in its reduced form as a hydroquinone (ubiquinol) and is a potent lipophilic anti-oxidant that has great importance as a free radical scavenger. It protects the stability of the cell membranes, protects DNA from free radical-induced oxidative damage, and is capable of recycling and regenerating other anti-oxidants such as tocopherol and ascorbate.5 The intracellular synthesis of CoQ10 constitutes the main source in humans but CoQ10 is also found in small amounts in a wide variety of foods. It increases in human skin from childhood to maturity, then decreases with age and irradiation from UVA rays, which compromise the skin’s anti-oxidant balance, leading to increased Reactive Oxygen Species (ROS) concentration in aged skin.6
Moreover, the epidermis contains a tenfold higher level of CoQ10 than the dermis. As the outermost layer of skin, the epidermis is directly exposed to UV irradiation, which is known to deplete antioxidants such as CoQ10.7, 8 Interestingly, CoQ10 has been demonstrated to directly eliminate free radicals in elderly subjects.9 Further, the reduced form of ubiquinone, or ubiquinol (see Figure 2), can rescue tocopheryl radicals produced by reactions with lipid or oxygen radicals by reverting them back to tocopherol.10
For all these reasons, anti-oxidant protection should be of great interest with respect to skin aging, and accordingly, CoQ10 may be an ideal target to preserve skin. Compound IV08.004 was therefore evaluated in vitro for its ability to help skin supply CoQ10 at the cellular level, as described here. In addition, the activities of the compound to target PDSS1/2 enzyme an dboost CoQ10B protein, the natural, chaperone, were tested in vitro and ex vivo. Further, its actions to visibly reduce the appearance of wrinkles and fine lines were assessed in a clinical study.
Materials and Methods
Cells and skin biopsies: Human adult low calcium temperature (HaCaT) keratinocytes from immortalized aneuploid keratinocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM), 4.5 g/L glucose supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 0.1 mg/mL of an antiobioticb. Normal Human Keratinocytes (NHK) were cultured in keratinocyte serum free medium supplemented with 50 μg/mL bovine pituitary extract, 5 ng/mL human recombinant EGF and 0.1 mg/mL antibioticb. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Skin biopsy samples were obtained from a 61 year-old female European donor after abdominal plastic surgery. For histological experiments, the skin organ culture method was followed.
HPLC study of intracellular CoQ10: HaCaT Cells were treated twice daily with 1% of the biofunctional compound IV08.004 for 24 hr. Cells were washed with PBS and detached with trypsin EDTA 200 mg/L for 10 min at 37°C, 5% CO2. Cells were collected and centrifugated, the supernatant was discarded and the pellet of cells was washed with distilled H2O and dissolved in 700 μL of mixture hexane/isopropyl alcohol (IPA) (5:2) and underwent three freezing/thawing cycles, sonication (10 sec, 130 watt, 20 KHz) and centrifugated again at 4°C for 10 min. The supernatant was collected and evaporated. After total evaporation, 200 μL of IPA was added and high pressure liquid chromatography (HPLC) assay was performed.
Detection of CoQ10B and/or UVB Stress: NHK were treated twice daily with 1% of compound IV08.004 for 48 hr with 1% of compound IV08.004. Skin biopsies were treated twice within 24 hr with a 20-μL application of either 1X PBS as the control condition, or with 1% of IV08.004 diluted in PBS on the top of the biopsies. Biopsies were submitted to 100 mJ/cm² of UVB in a UV oven to ensure homogeneity of the irradiation and again treated twice within 24 hr. At the end of the experiment, tissues and cells were prepared for immuno-staining as described below.
Paraffin-embedded preparation: NHK were fixed with 10% neutral formalin for 1 hr then centrifuged for 5 min at 2,000 rpm. Cells and biopsies were paraffin-embedded in an automated device, prepared using a cell block preparation systemc, cut into 4-μm thick sections with a microtome and collected on poly-lysinated slides for immuno-staining.
Immunofluorescent detection of CoQ10B: 4-μm sections of paraffin-embedded NHK or skin sections were deparaffined in 100% xylene and rehydrated in graded alcohols. Sections were incubated with 5% BSA for 30 min, then with a rabbit polyclonal anti-CoQ10 binding protein B diluted in PBS for 1.5 hr under agitation at room temperature (RT). After extensive washing in PBS, fluorescent secondary antibodyd was applied for 1 hr at RT. Nuclei were stained with a 0.3 μM 4', 6’-diamidino-2-phenylindole (DAPI). Samples were washed for 5 min in PBS, CoQ10 binding protein B detection was managed and examined using a microscope with 20x or 40x objectives, and photos were captured.
ROS detection in keratinocytes after UVA exposure: NHK were maintained in culture at 37°C under 5% CO2 in a humidified atmosphere, for 24 hr with or without one application per day of 0.5% compound IV08.004 directly in the medium. After 24 hr of culture, the medium was removed and the cells were irradiated at 4 J/cm² of UVA in 1X PBS. Then, cells were incubated for 1 hr more at 37°C. Cell medium was removed and 5 μM of mitochondrial superoxide indicator solutione was added on cells for 10 min at 37°C. Cells were washed three times with 1x PBS and fixed with 3.7% formaldehyde for 10 min at RT under gentle agitation in a dark room. Nuclei were stained with a 1:1000 dilution of 0.3 μM DAPI in 1x PBS for 5 min. The microscopic observations were carried out using a microscope with a 40x objective and photos were taken.
Anti-aging clinical evaluation on 10 volunteers: A 28-day, double-blind study was conducted on 10 volunteers versus placebo. Volunteers applied a test and placebo cream (proprietary) twice daily on the crow’s feet area (2 mg/cm²) and a silicon replica was performed at days 0 and 28 after treatment. The negative replica was assayed by softwaref, and roughness parameters were calculated. Briefly, calculations included total roughness (Rt), maximal roughness (Rm), depth of roughness (Rd), average of five successive segments (Rz), volume, and the surface of skin’s micro-relief.
Quantification and statistical analysis: Three or four images per condition were converted to grey levels and quantified with softwareg. The sum of pixel numbers per intensity was calculated and adjusted by considering comparable areas or numbers of cells.11 For in vitro tests, statistical analysis was conducted using an unpaired student t-test with an unilateral distribution; p < 0.05 was considered significant, p < 0.01 as very significant, and p < 0.005 as highly significant.
Results and Discussion
CoQ10 increase in HaCaT cells: The HPLC chromatogram of extracts from untreated and 1% compound IV08.004-treated HaCaT showed a selective elution peak corresponding to CoQ10. The measurement showed an increase in CoQ10 expression inside the cells by 46% after 24 hr (see Figure 3).
CoQ10 binding protein increase: Immunofluorescence detections showed an increase in CoQ10B expression both in 1% compound IV08.004-treated keratinocytes and human skin biopsies; i.e., +272% and +83%, respectively, compared with the untreated condition after 48 hr of culture (see Figure 4 and Figure 5). This large increase of CoQ10B in cells may be due to the technical approach used. Parafin-embedded-cell blocks, i.e., pellets, increase cell number density; therefore, this technical approach to cut cell pellets in thin slices may highlight more immunological sites than would cells in a classical immunological staining.
Skin biopsies pre-treated with 1% compound IV08.004 and submitted to UVB radiation displayed a significant 44% enhancement in CoQ10B expression, compared with the untreated control, whereas untreated biopsies submitted to UVB radiation showed a 55% decrease in CoQ10B expression. Results indicated the compound limited the decrease in CoQ10B expression, highlighting a possible role of CoQ10B in protecting the skin from oxidative stress (see Figure 6).
Limiting ROS formation after UVA exposure: As noted, mitochondrial indicator was used to assess superoxide production in the mitochondria of cultured cells. When NHK were exposed to UVA irradiation, there was an excessive 440% induction of ROS, compared with unstressed cells. Treatment using 0.5% compound IV08.004 for 24 hr partially prevented ROS production, i.e., 234% vs. 440%; indeed, it seems to have limited the production of ROS in cells by more than half (see Figure 7).
Clinical evaluation: The in vivo effects of compound IV08.004 were determined after 28 days of application in a double-blind clinical study assessing the classical parameters of roughness. The side treated with compound IV08.004 showed a decrease in all parameters compared to placebo, and these measurements were significant according to the Wilcoxon test; moreover, the benefit of the formula was visible on photographs of the crow’s feet (see Figure 8).
In this work, the authors present a new compound based on pentapeptide-34 trifluoroacetate, referred to as IV08.004, that targets CoQ10 binding proteins and may contribute to a boost in CoQ10 in the skin. The in vitro, ex vivo and clinical efficacy of IV08.004 were summarized.
First, chromatography HPLC showed that it enhanced the level of CoQ10 by 46% in skin cells. Also, the ability of IV08.004 to significantly increase CoQ10-binding protein expression was demonstrated both in keratinocytes (272%) and human skin biopsies (83%). CoQ10 binding protein is required for the function of Coenzyme Q10 and may serve as a chaperone or to transport from its site of synthesis to the catalytic sites of respiratory complexes. Thus, the efficacy of the compound to increase the expression of CoQ10 binding protein is strongly correlated with the presence of CoQ10 itself.
Indeed, this is exactly what was observed in HaCaT cell experiments, where the amount of Coenzyme Q10 in cells increased by 46% after treatment with the compound. Further, bibliographic data supports the claim that CoQ10 is a potent lipophilic anti-oxidant that acts as a ROS scavenger.12 Similarly, the authors showed here that increased ROS production in the mitochondria from UVA irradiation decreased by 38% when pre-treated with 0.5% of compound IV08.004. Moreover, this experiment was performed with other stressors including UVB and rotenone (data not shown), and the same results were observed; under irradiation or during aging, the ROS concentration in skin increased and the amount of CoQ10 decreased.6 Therefore, this in vitro study suggests that compound IV08.004 is a potent antioxidant that may help to preserve skin against oxidative damage.
While intracellular synthesis is the major source of CoQ10 in humans, it is not the only one. CoQ10 is found in small amounts in a variety of food from animal sources. Moreover, CoQ10 is popular as a freely available dietary supplement. Nevertheless, little relevant research exists examining its appropriate dose and, above all, its bio-availability. It is worth noting that exogenous CoQ10 is always in the oxidized form and is unable to provide antioxidant properties; it would require enzymes such as diaphorase and lipoamide dehydrogenase to convert it to a reduced form.13
Numerous topical formulations already exist in the cosmetic market that contain CoQ10 and claim to possess anti-oxidant, skin repair and regeneration, anti-wrinkle and anti-aging capabilities, but generally the efficacy remains low. Compound IV08.004 offers a new approach to address these concerns in skin. Clinical tests demonstrated the effectiveness of the compound to decrease wrinkles and fine lines after 28 days of application, in comparison with the same formula excluding the test ingredient. In addition, a statistically significant improvement in skin roughness parameters was demonstrated with use of the ingredient. Thus, as CoQ10 is an integral part of the mitochondrial respiratory chain, supplementation with CoQ10 may contribute to balanced cellular energy metabolism at an advanced age. The studies described here indicate that the modulation of CoQ10B expression seems to protect skin from oxidative damage, confirming the industry’s interest in enhancing CoQ10 in skin for anti-aging effects.
Acknowledgements: The authors wish to thank Zelmira Lazarova for generously providing the HaCaT cell line.
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