Rhythmic activity appears to be a fundamental property of living matter and the activities of cells obey daily or seasonal periodicities.
Introduction
The cutaneous biological clock
Rhythmic activity appears to be a fundamental property of living matter and the activities of cells obey daily or seasonal periodicities.
The circadian rhythms regulating the mitoses of keratinocytes and fibroblasts were discovered in the course of studies on rodents following initial observations that cell responses differed depending on the time at which epidermal growth factor (EGF) was injected into mice (Yeh, 1981). Since then, numerous studies have shown that the daytime/night-time variations in EGF secretion and membrane expression of EGF receptor (EGFr) control and regulate cell proliferation in numerous epithelial tissues, including the skin (Bjarnason, 2002).
Thanks to the work of a few teams (Tsukahara H, 2001; Zanello, 2000; Lefur 2001), we are now aware that certain cutaneous functions are preferentially expressed at night: higher values for the microcirculation, trans-epidermal water loss (TEWL) and acid phosphatase; others, on the contrary, are more marked during the day: sebaceous secretion, pH, reaction to allergens.
During the day, the skin protects itself against aggressors; during the night, it renews itself. Thus, Brown (1991), reported an increase in keratinocytic mitoses at night
(11.30 pm) while the DNA multiplication phase peaked at 3.50 pm. Information on the EGF acrophase in various organs is lacking. However, an article by Ino (1993) reported a peak salivary EGF concentration between 10 pm and 7 am, during the resting period.
This suggests that DNA synthesis and the mitoses of keratinocytes are correlated with the day/night periodicity of EGF and its receptor.
Cell stimulation by phosphoinositides
Calcium is known to play a preponderant role in cell metabolism: the cell has an intracellular pathway for releasing the calcium stored in the reticulum. The latter contributes to ‘waking up the cell’. A cell at rest maintains very low levels of free calcium in the cytosol, thanks to a pump that continuously expels calcium towards the exterior and towards the endoplasmic reticulum (Ca++-ATPase).
The mobilization of intracellular Ca++ is actively related to phosphoinositides such as phosphatidylinositol (PI), phosphatidylinositol diphosphate (PIP2) and inositol triphosphate (IP3). The latter is a key molecule and messenger that leads to intracellular calcium ion release but is also a precursor to the synthesis of IP4, IP5, IP6 and further polyphosphorylated analogs which constitute an energy reserve that can be mobilized at any time by kinases and, in particular, are used to feed the ATP pool (Voglmaier, 1996).
The phosphoinositide molecules, known as ‘second messengers’, can trigger numerous intracellular mechanisms since they provide both energy in the form of their phosphorylated groups, and the diacylglycerol skeleton (PKC-dependent activations). The importance of those pathways is abundantly illustrated in the literature: Ca++ and IP3 are necessary for fibroblast-collagen interactions (Ahlen, 1998, Payrastre, 1991, Igwe, 1997, De Boland, 1996).
The hypothesis that keratinocyte DNA synthesis and mitoses are correlated with the day/night periodicity of EGF and its receptor is supported by the work of Payrastre (1991) who established the close structural and functional relationship between the phosphoinositide transduction cascade and EGF.
Euglena and day/night cycles
Euglena (Euglena gracilis) is a unicellular microalgae-like protist that naturally lives in fresh water. Its metabolism is strongly dependent on the presence or absence of light.
During the day, Euglena synthesizes chlorophyll in large quantities. At night, it becomes rich in precursor carotenoids and reserve energy molecules such as phospholipids (phosphatidylinositol, phosphatidylethanolamine, phospha-tidylcholine, phosphatidyl-l-glycerol) and in penta- and hexakisphosphates which constitute a storage form of phosphoinositides and a very rich energy donor enabling restoration of the ATP pool.
Those membrane lipids account for about 20% of the dry weight of the cell wall of Euglena and help it to adapt conformationally and metabolically to marked changes in its living conditions (Nakano, 1987 and Miller, 1978). The Euglena cell, like skin cells, is one whose growth is modulated by the day/
night alternation. Euglena’s cytoskeleton is a complex matrix affording all the components involved in the acquisition (receptors) and processing of the circadian signal (phospholipids and phosphoinositide cascade). Once again, the key point
is the importance of phosphoinositides in cell activation and their clear link with cell rhythms.
The cosmetic value of the original Euglena membrane lipid composition consists in the fact that it is a potential source of functional precursors to stimulate the skin cells by mechanisms shared by Euglena and human skin cells. We thus developed CHRONODYN™, an Euglena extract, as a source of cell vitality and chronobiological stimulation and tested it for cosmetic activity and use.
Demonstration of the cosmetic
properties of CHRONODYN™
In vitro studies
de novo IP3 synthesis in keratinocytes
Human keratinocytes were cultured and incubated to 90% confluence. The cells were incubated in the presence of Chronodyn at various concentrations, or the control, for
1 minute. The IP3 produced by the cell was extracted with perchloric acid and assayed by an RIA method (Amersham kit). The positive control consisted of Platelet-Activating Factor (PAF). All assays were repeated in triplicate.
Results
As expected, PAF stimulated IP3 production. The stimulant effect of Chronodyn is clearly observed with a dose-effect: the de novo synthesized IP3 pool was markedly increased. At a concentration of 3% Chronodyn, the keratinocytes produced three times as much IP3.
These results demonstrate that Chronodyn is a potent cell stimulator whose mode of action via the phosphoinositide pathway results in an increase in the intracellular IP3 pool.
Calcium flash triggering in keratinocytes
Keratinocytes were cultured to confluence. A fluorescent probe (Fluo-4Am) whose fluorescence is proportional to the quantity of free intracellular calcium was added to the culture medium for 15 minutes. The cells were then rinsed and the products were tested in comparison with control cells and in comparison with Platelet-Activating Factor (PAF). The cells respond to IP3 stimulus by releasing calcium from the reticulum into the cytosol. Video recording enables visual monitoring of the release kinetics.
Results
In Figure 5, the image on the left shows T0 before product addition. The image on the right was obtained after a few seconds of contact with 3% Chronodyn.
As expected, PAF (Platelet-Activating Factor) induced a calcium flash after a few seconds of contact with the cells. Exposure of the cells to Chronodyn also resulted in a calcium flash whose intensity was proportional to Chronodyn concentration. These results fully corroborate those obtained for de novo IP3 synthesis. Thus, IP3 production and the calcium flash are closely linked in the cell activation cascade.
de novo ATP synthesis
Normal human fibroblasts were cultured in classic DMEM medium, and then deprived of ATP by addition of deoxyglucose (a decoy which inhibits ATP synthesis via the Krebs cycle). Following the depletion phase, deoxyglucose is eliminated by washing and replaced by normal culture medium or culture medium enriched with varying concentrations of Chronodyn. The quantity of intracellular ATP is determined by bioluminescence through coupling with a luciferase/luciferin system. The luciferin formed by luciferase in the presence of ATP emits luminescence proportional to the quantity of ATP present. The quantity of ATP synthesized in 2 hours is measured and hence a restoration rate is determined.
Results
A clear dose-dependent increase in intracellular ATP levels is observed, reaching +48% after 2 hours of incubation in the presence of 3% Chronodyn.
The increase in the ATP pool, the de novo IP3 synthesis and the calcium flash obtained when keratinocytes were incubated in the presence of Chronodyn altogether validate Euglena extract as a source of cell vitality and energy and provide hope for cutaneous improvement through enhanced cellular response to chronobiological messengers.
In vivo studies
Chronodyn is designed to stimulate cutaneous vitality and energy. The parameters of skin resistance to torque, ‘deformability’, and ‘recovery’ (i.e. return to the basal state after torsion) were monitored in the course of this study.
A double blind vehicle controlled efficacy study was conducted on a group of 21 women of mean age 44 years (40 to 49 years) with moderate sagging of the cheeks. Usual inclusion and exclusion criteria were used. The volunteers applied a cream containing 3% Chronodyn to one side of the face twice daily and the vehicle cream to the other side. Each subject thus acted as her own control. Measurements of skin firmness were conducted on T0, T14 and T28 days. The dermal torque meter (Diastron) was used for the study. One site was chosen on each cheek, precisely located for each point in time (T0, T14 and T28). The duration of torsion (15 mNm) was 5 seconds followed by a 5 second relaxation period. The determinations were recorded after 1 torsion and after 5 consecutive torsions. The deformability measurements are considered to reflect cutaneous vitality while the recovery determinations demonstrate resistance to cutaneous fatigue and thus a more energetic skin. The parameters monitored were: the deformability, ‘Ue’, and the residual extension, ‘r’, after 1 and 5 torsions.
Results
The creams containing Chronodyn were very well tolerated by all the volunteers.
Cutaneous vitality and deformability (Ue)
A product increasing cutaneous vitality is expected to decrease deformability of the skin subjected to torque.
As expected, the results (Table 2) show a decrease in deformability when the skin is treated with Chronodyn. The improvement was even more marked for the parameter (Ue) after 5 consecutive torsions since there was a significant decrease in deformability of 21.3% after 28 days of application. Over the same time period, the skin treated with the vehicle cream showed an increase in cutaneous deformation both over time and with the number of torsions: the skin grew tired and recovery was less effective.
The contrasting results obtained with Chronodyn and the vehicle leave no doubt as to the beneficial effect of procured by Eurglena extract.
Cutaneous energy and recovery duration (r)
An energizing product is expected to decrease the recovery time parameter.
Again, the results show the expected decrease in the residual deformation, i.e. the recovery time parameter (r) of the Chronodyn treated skin (Figure 6).
A significant decrease was observed after 14 days; the improvement was even more marked after 28 days, (-33.5%): the skin resists more energetically and ‘tires’ less rapidly. This is particularly true after 5 consecutive torsions.
Over the same period, the vehicle cream showed an increase in the deformation and a lack of tonicity of the skin: the skin had lost some of its ability to recover. These results are illustrated in the Figure 6.
Conclusion
Chronodyn is a standardised extract of Euglena gracilis, a unicellular aquatic organism that is particularly rich in phosphoinositides. Phosphoinositides are also normally present in mammalian cells, particularly those of the cutaneous tissue where they contribute to activating cell metabolism.
Chronodyn has been shown to provide skin cells with an energy supplement by stimulating ATP recovery, IP3 synthesis and calcium release in keratinocytes. This translates into a significant decrease in cutaneous deformability and recovery duration, as was demonstrated in a 28 day clinical trial.
All these results document Chronodyn as a potent cell stimulant able to give tired and aged skin cells the energy they are lacking, as is the case with ageing cells.