Several research studies on aged people carried out by different research teams have highlighted the existence of a new theory in skin ageing named ‘inflammaging’.1–3 Skin inflammaging results from a combination of several deleterious pathways inducing a vicious cycle of micro-inflammation.
A cascade of inflammatory responses results in chronic low level inflammation and leads to accumulation of molecular and cellular damages, well-known sources of skin ageing which can affect the appearance of the skin. Laboratoires Sérobiologiques (LS) – now with Beauty Creations – developed the first cosmetic active – a bark extract from Eperua falcata – which targets inflammaging in a comprehensive way, by addressing the consequences of its deleterious effects on the skin in a particularly efficient manner.
Ageing results from a chronic asymptomatic micro-inflammatory state, differing from acute inflammation. During the ageing process, skin is exposed to numerous pro-oxidant and pro-inflammatory insults, and a continuous up-regulation of proinflammatory mediators (TNF, IL-1, IL-6, IL-8) is induced.3,4 This elevation of circulating pro-inflammatory messengers induces the release of reactive oxygen species and molecular damage to nucleic acids, polysaccharides, proteins and lipids, which in turn activates several micro-inflammatory pathways maintaining and aggravating the process. This vicious micro-inflammatory cycle is a major factor underlying ageing and potentially many age-related diseases such as Alzheimer’s disease or arthritis.2,3 In the skin, some pathways are particularly involved in the establishment and maintenance of this vicious microinflammatory cycle (Fig. 1):
• The NF-B pathway, inducing pro-inflammatory cytokine release5 The Nuclear Factor-kappa B (NF-B) is a pro-inflammatory transcription factor for the host defence. It is involved in cellular responses to a wide array of external and internal stimuli such as cytokines, free radicals, DNA damage, UV, immune attacks. Upon activation, it induces the transcription of a large number of genes of the inflammatory responses, including cytokines, adhesion molecules and other immunoregulatory proteins. NF-B is an important target to fight against inflammaging, as it has been shown to be constitutively activated with ageing. In resting cells, the NF-B complex is kept inactive. Upon stimulation, it is translocated in the nucleus wherein it activates gene transcription of proinflammatory cytokines such as IL-8. • The release of neuropeptides by terminal nerve endings, involved in neurogenic inflammation6,7 The skin includes many nerve fibres, in particular the C fibres present in the epidermis up to the stratum corneum. They convey input signals from the peripheral to the central nervous system, and by the local release of pro-inflammatory neuromediators are associated with the development of neurogenic inflammation. The stimulation of these cutaneous nerve endings by several stimuli (environmental or chemical) locally provokes the release of many neuropeptides like CGRP in the skin activating the release of cytokines (IL-1, TNF, PGE2), inducing a vasodilation and an inflammatory process, which in turn potentiates the neuronal response. • The plasmin protease, involved in skin barrier disruption8 Plasmin is a serine protease formed in human epidermis from the activation of plasminogen by urokinase-type Plasminogen Activator (uPA), a protease expressed at the cell surface of basal keratinocytes. Pro-inflammatory cytokines IL-1and TNFinduce an increase of uPA expression and of plasmin mediated proteolysis in human epidermal keratinocytes.9 Plasmin activity in the stratum corneum is involved in chronic micro-inflammation and compromised skin barrier function,8 with positive correlation between plasmin activity and increasing levels of TEWL. Such a disruption of the stratum corneum barrier not only is characteristic of many inflammatory conditions, but induces inflammation itself. The micro-inflammatory state resulting from the activation of these three pathways accelerates ageing by inducing the release of reactive oxygen species and molecular damage, thus leading to a loss of tonicity, elasticity and firmness as well as a compromised skin barrier function. Eperua falcata extract [INCI name: Maltodextrin (and) Eperua Falcata Bark Extract] is the first ingredient which targets inflammaging in a comprehensive way by the modulation of these micro- inflammatory pathways and by addressing the consequences of its deleterious effects on the skin. Also, the raw material conforms to Ecocert standards of Natural and Organic cosmetics. A range of tests have been performed to demonstrate in vitro its efficacy on the inhibition of TNF-induced NF-B activation and cytokine release, of the release of pro-inflammatory neuropeptides and of the plasmin activity, and in vivo on the improvement of skin biomechanical properties and skin barrier function.
Materials and methods
TNF-induced NF-B nuclear translocation on human keratinocytes (in vitro test)
Normal human epidermal keratinocytes were isolated from plastic surgery of adult donors. At subconfluence, the products were introduced in new culture medium at different doses. The positive reference is NF-B Activation Inhibitor III at 10 M. The cells were incubated with the products during 24 hours (2 hours for the positive reference) at 37°C, CO2 = 5%, before addition of TNFat 5 ng/mL. After 20 minutes of incubation, the cells were washed, fixed and permeabilised. The level of NF-B in the nucleus was measured by immuno-fluorescence, whereas the cell nuclei were stained with Hoechst 33258. The cell viability was previously determined through the MTT-test in order to exclude cytotoxic doses.
TNF-induced IL-8 release on human keratinocytes (in vitro test)
Normal human epidermal keratinocytes were obtained as previously described. At cell confluence, the products were introduced in fresh culture medium at different doses with TNFat 100 ng/mL. The positive reference is hydrocortisone at 1 M. After a subsequent incubation of 24 hours at 37°C, CO2 = 5%, the quantity of IL-8 in culture supernatant was evaluated by ELISA kit. The cell viability was previously determined through the MTT-test in order to exclude cytotoxic doses.
CGRP release from cultured sensory neurons (in vitro test)
The level of CGRP in culture supernatant was evaluated by ELISA on cultured sensory neurons (differentiated dorsal root ganglia) after incubation for 20 minutes at 37°C and 5% CO2, and stinging during 25 minutes by capsaicin at 1 M, or membrane depolarisation by KCl at 40 mM. Lack of cytotoxicity was previously determined by MTT-test.
Plasmin inhibition (in vitro test)
Human plasmin activity inhibition was evaluated by degradation of labelled casein as substrate. Products, human plasmin and labelled casein were diluted and mixed in phosphate buffer pH 7.4. The positive reference is soybean trypsin inhibitor (SBTI) at 1 M. Plasmin-catalysed hydrolysis of labelled casein relieves the quenched fluorescence. The rate of hydrolysed substrate was determined by recording this fluorescence (excitation at 485 nm and emission at 538 nm) at 0 and 30 minutes of incubation at room temperature.
Improvement of skin biomechanical properties and barrier function (in vivo test)
A randomised vehicle-controlled, home-inuse study was carried out on 52 volunteers between 30 and 65 years old (mean age±SD = 54±1) in order to evaluate the effects of the topical application of a formulation containing 1% of Eperua falcata extract. The O/W emulsion containing the active ingredient and the placebo-vehicle were applied by the volunteers twice a day on the hemi faces, under normal use conditions. The measurement of biomechanical properties (temples) with Cutometer SEM 575 (Courage & Khazaka) and the TEWL (upper cheek) with Aquaflux AF100 (Biox Systems Ltd) was done before the first application of the product and at the end of 28 days of treatment.
Results and discussion
Inhibition of NF-B pathway and IL-8 release (in vitro test on human keratinocytes)
Eperua falcata extract displayed a significant dose-dependent inhibition of TNF-induced nuclear translocation of NF-B, up to 67% of inhibition at 0.1% (Fig. 2, activated NF-B in nucleus). This effect on the NF-B signalling pathway was confirmed through the inhibition of IL-8 release from TNFtreated human keratinocytes. The extract exhibited a significant dosedependent anti-inflammatory effect on IL-8 release, up to 77% of inhibition (Fig 3).
Inhibition of release of proinflammatory neuropeptide CGRP by sensory neurons (in vitro test)
The sensory neurons in culture were stimulated by membrane depolarisation using KCl, or by the TRPV1 agonist capsaicin, leading to an increase of CGRP released in the culture medium. At non cytotoxic doses, Eperua falcata extract displayed a dose-dependent inhibition of released CGRP, up to 56%-60% at 0.033% (Fig. 4).
Inhibition of plasmin (in vitro test)
Eperua falcata extract dose-dependently inhibited the plasmin activity with an IC50 (inhibitory concentration 50%) of 0.02% (Fig. 5).
Protection against environmental and oxidative stresses (in vitro test, data not shown)
In addition, the capacity of Eperua falcata extract to protect epidermal cells against the environmental and oxidative stresses aggravating this micro-inflammatory vicious cycle was further assessed in different in vitro tests. Eperua falcata extract inhibited the release of superoxide anions, the UVAinduced oxidation of cell membrane lipids on fibroblasts, and the cytokines induction in UVB-irradiated keratinocytes (data not shown).
This chronic micro-inflammation in skin induces molecular damages and decreases the ability of the skin to repair itself, leading to a degradation of the skin biomechanical properties, and a compromised skin barrier function. In the clinical study, the improvement of these two visible signs was evaluated on the face of women volunteers with an emulsion containing 1% of Eperua falcata extract, comparatively to the placebo emulsion. The effect on biomechanical properties was evaluated using Cutometer by the measurement of the elastic deformation (Ur/Ue), the elastic recovery (Ur/Uf), and total deformation ratio (Ua/Uf), which have been shown to be negatively correlated with age10 and sagging severity.11 The reinforcement of skin barrier function was determined by measurement of TEWL (Trans Epidermal Water Loss). The 28 days of treatment with the emulsion containing Eperua falcata extract significantly improved both the elastic deformation (Ur/Ue), the elastic recovery (Ur/Uf) and total deformation ratio (Ua/Uf) (p value = 0.0131; 0.0209 and 0.0068 respectively for Ur/Ue, Ur/Uf and Ua/Uf). The increase of these three ratio parameters was between 10% and 12% comparatively to baseline, whereas the placebo emulsion displayed only nonsignificant 1%-4% modification of these ratios (Fig. 6). Comparison between placebo and Eperua falcata extract treated sites is very close to significance with a p value of 0.058 and 0.084 for Ur/Ue and Ur/Uf respectively. The treatment of the skin with tested emulsions has reduced the TEWL (Fig. 7). While this improvement was not significant for placebo treatment, it was significant (p=0.0064) for the emulsion containing Eperua falcata extract and reached 10% over the 28 days of treatment. After 28 days of treatment, the cream containing 1% Eperua falcata extract has shown significant restorative effects against age-related degradation of skin biomechanical properties, loss of elasticity, firmness and tonicity involved in skin sagging. In addition, the Eperua falcata extract containing formulation has reinforced the skin barrier integrity, improving the ability of the skin to act as a physical and mechanical barrier against exogenous factors.
Skin inflammaging results from a combination of several deleterious pathways inducing a vicious cycle of microinflammation. Our results showed that Eperua falcata extract is able to inhibit in vitro this vicious cycle induced by this chronic low grade upregulation of the inflammatory response. Moreover Eperua falcata extract proved to be effective in vivo: improvement of the age-related loss of elasticity, firmness and tonicity, reinforcing the skin barrier function and thus minimising these visible signs of skin ageing.
1 Franceschi C et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 2000; 908: 244-54. 2 Bruunsgaard H et al. Age-related inflammatory cytokines and disease. Immunol Allergy Clin North Am 2003; 23: 15-39 3 Chung HY et al. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev 2009; 8: 18-30. 4 Mariani E et al. Simultaneous evaluation of circulating chemokine and cytokine profiles in elderly subjects by multiplex technology: relationship with zinc status. Biogerontology 2006; 7: 449-59 5 Salminen A et al. Genetics vs. entropy: longevity factors suppress the NF-B driven entropic ageing process. Ageing Res Rev 2010; 9: 298-314. 6 Peters EMJ et al. Neuropeptide control mechanism in cutaneous biology: physiological and clinical significance. J Invest Dermatol 2006; 126: 1937-47. 7 Rossi R et al. Cutaneous innervation and the role of neuronal peptides in cutaneous inflammation: a review. Eur J Dermatol 1998; 8 (5): 299-306. 8 Voegeli R et al. Increased basal transepidermal water loss leads to elevation of some but not all stratum corneum serine proteases. Int J Cosmet Sci 2008; 30: 435-42. 9 Bechtel MJ et al. Upregulation of cell-surfaceassociated plasminogen activation in cultured keratinocytes by interleukin-1and tumor necrosis factor-. Exp Cell Res 1996; 223: 395-404. 10 Krueger N et al. Age-related changes in skin mechanical properties: a quantitative evaluation of 120 female subjects. Skin Res Technol 2011; 17: 141-8. 11 Ezure T et al. (2010) Influence of subcutaneous adipose tissue mass on dermal elasticity and sagging severity in lower cheek. Skin Res Technol; 16: 332-8
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