Eperuline. by Beauty Creations. Inflammaging: the new theory in skin aging

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1 Eperuline by Beauty Creations Inflammaging: the new theory in skin aging

2 What if all was not discovered on inflammation and aging? Inflammaging is a well known phenomenon in the medical field, e.g. in Alzheimer s disease or arthritis [1-4]. It is characterized by a chronic, low grade, asymptomatic micro-inflammatory state with elevated level of inflammatory markers, differing from acute inflammation. This up-regulation of the inflammatory response is a major factor underlying age-related diseases and aging. In cosmetics, inflammation (e.g. skin redness) and aging are mostly addressed independently and the converging inflammaging process is not substantiated as well as in the medical field. 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 damage, well-known sources of skin aging which can affect the appareance of the skin. Some pathways are particularly involved in the initiation and propagation of this vicious microinflammatory cycle: - the NF-kB pathway inducing cytokine release [5], - the inflammatory-related serine protease plasmin [6], - the release of pro-inflammatory neuropeptides by terminal nerve endings in the epidermis [7] (Fig.1). NF-κB Chronic microinflammation 1 Neuropeptides Plasmin Aging 2 3 Fig 1 - NF-kB, plasmin and neuropeptides are key factors of chronic low grade inflammation response and aging. Ultimately, the micro-inflammatory state resulting from the activation of the NF-kB pathway and cytokine release, plasmin activity, and release of neuropeptides, accelerates aging 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. EPERULINE PW is the first active to target inflammaging in a comprehensive way by consecutively addressing the consequences of its deleterious effects on the skin in a particularly efficient manner. The main objective of EPERULINE PW is to fight against chronic low grade inflammation and its visible consequences on skin aging, via 3 different pathways: 1 inhibition of the NF-kB activation and cytokine release: action against mediators of inflammation. 2 inhibition of plasmin serine protease: skin barrier protection. 3 inhibition of neuropeptides release by activated peripheral sensory nerve endings: action against neurogenic inflammation. The action on these 3 pathways completed with an anti-oxidative effect results in a perceivable improvement on skin biomechanical properties as well as a good protection of the epidermis by enhancing the skin barrier function. The visible effects of the age-related loss of elasticity, firmness and tonicity such as the ones involved in skin sagging, are delayed.

3 Definition / Composition EPERULINE PW is an aqueous extract from the bark of the tree Eperua falcata Aubl. This species is a dominant tree widely distributed in the Guyanese rainforest. The bark and resin are used traditionally by local Amerindians for toothache, wound healing or to ease joint pains. The wood is also used for carpentry purposes and the manufacture of rot proof poles. EPERULINE contains dihydroflavonols and catechuic tannins among other components. Skin benefits Improvement of barrier function. Protection against oxidative stress. Improvement of skin firmness. Fights sub-chronic microinflammation. Cosmetics use Anti-aging skin care: - anti-sagging - firming - tonicity improvement.

4 Efficacy tests The efficacy of EPERULINE PW LS 9627 against the vicious cycle of micro-inflammation and its visible consequences on skin aging was evaluated both in vitro and in vivo. In vitro studies were performed investigating the TNFa-induced NF-kB activation and cytokine release, the plasmin activity, the release of pro-inflammatory neuropeptides, and anti-oxidative efficacy. An in vivo study with a formulation containing EPERULINE PW was carried out to determine the improvement of skin biomechanical properties and of skin barrier function. Inhibition of NF-kB pathway and cytokine release (in vitro tests) Aim To evaluate on human keratinocytes the activity of EPERULINE PW against: - TNFa-induced NF-kB nuclear translocation and IL-8 release, - UVB-induced expression of cytokines. Background The NF-kB signaling pathway plays a central role in the micro-inflammatory aging process [5]. The NF-kB system is specialized in the host defense against infectious agents and environmental and cellular stress. In resting cells, the NF-kB transcription factors are kept inactive in the cytoplasm by association to several inhibitory IkB proteins. Upon cell stimulation, several protein kinases phosphorylate the IkB proteins, triggering the translocation of NF-kB complexes to the nucleus where they can activate gene transcription, especially the inflammatory genes. During aging, persistently elevated NF-kB activation induces the expression of proinflammatory chemokines and cytokines, such as IL-8, that trigger chronic low grade inflammation. This NF-kB activation is mediated by different age-related factors such as redox imbalance, DNA damage, activation of innate immunity, TNF superfamily cytokines, which are impacted by extrinsic environmental factors such as UV-irradiation, pollution and stress. Protocol Culture of human keratinocytes in growth medium Incubation for 3 days at 37 C, CO 2 Exchange of growth medium by medium containing EPERULINE PW (*) (NF-κB Activation Inhibitor III used as positive control) Incubation for 1 day at 37 C, CO 2 Addition of TNFα and incubation for 2 minutes at 37 C Visualization of NF-κB by immunocytochemistry (ICC) and quantification of NF-κB translocated in nucleus of stimulated cells Fig. 2 Schema of protocol: TNFa-induced NF-kB nuclear translocation. (*) Lack of cytotoxicity of EPERULINE PW was preliminary tested by an MTT test. Culture of human keratinocytes in growth medium Incubation for 3 days at 37 C, CO 2 Exchange of growth medium by medium containing TNFα and EPERULINE PW (*) (hydrocortisone at 1 µm used as positive control) Incubation for 1 day at 37 C, CO 2 Quantification of released IL-8 by an ELISA method Fig. 3 Schema of protocol: TNFa-induced IL-8 release. (*) Lack of cytotoxicity of EPERULINE PW was preliminary tested by an MTT test. Culture of human keratinocytes in growth medium Incubation for 3 days at 37 C, CO 2 Exchange of growth medium by balanced salt solution containing EPERULINE PW then UVB irradiation (5 mj/cm 2 ) (aspirine used as positive control) Incubation for 1 day at 37 C, CO 2 Quantification of released cytokines by a protein array Fig. 4 Schema of protocol: UVB-induced cytokines expression.

5 Results Control TNFa TNFa + EPERULINE PW at.1% Rate of NF-κB nuclear translocation in % / TNFα % inhibition Control TNFα TNFα NF-κB TNFα + Activation Eperuline TM PW Inhibitor III at 1 µm (%) * 72 n=3 Student's t test / TNFα *: p<.5 : p<.1 Fig. 5 Inhibition of TNFa-induced NF-kB nuclear translocation on human keratinocytes. 5 Rate of IL-8 in % / TNFα 1 6 * 58 Control TNFα TNFα hydrocortisone TNFα + at 1 µm Eperuline TM PW(%) * 66 * 53 * 23 77% inhibition n=6 ANOVA test PLSD of Fisher / TNFα *: p<.1 Fig. 6 Inhibition of TNFa-induced IL-8 release on human keratinocytes. Control Cytokine expression in % / UVB (n=2) UVB (5 mj/cm²) UVB + Aspirin at.1% UVB + UVB + EPERULINE EPERULINE PW at.3% PW at.1% Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD IL-1a 14 ± 3 ± ± ± 6 29 ± 6 IL-1b 38 ± 4 ± ± 27 8 ± 7 19 ± 4 IL-6 ± ± ± ± 1 17 ± 3 TNFa 9 ± ± ± 18 ± 64 ± 9 Fig. 7 Inhibition of UVB-induced cytokines release on human keratinocytes. Conclusion EPERULINE PW showed a significant dose-dependent inhibition of TNFa-induced NF-kB nuclear translocation and IL-8 release on human keratinocytes. The inhibitory effect of EPERULINE PW on the vicious cycle of microinflammation was confirmed on cytokine (IL-1a, IL-1b, IL-6 and TNFa) induction in UVB-irradiated keratinocytes.

6 Inhibition of plasmin (in vitro test) Aim To evaluate the inhibitory activity of EPERULINE PW on human plasmin. Background 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-1b and TNFa induce an increase of upa expression and of plasmin mediated proteolysis in human epidermal keratinocytes [8]. Plasmin activity in the stratum corneum is involved in chronic micro-inflammation and compromised skin barrier function [6, 9]. The urokinase-type plasminogen activator has been reported to be activated by barrier damage, and positive correlation was found between plasmin activity and increasing levels of TEWL [6]. Such a disruption of the stratum corneum barrier is not only characteristic of many inflammatory conditions, but induces inflammation itself. Moreover, plasmin degrades the components of basement membrane laminins, collagens type IV and VII, thereby impairing the assembly of the dermo-epidermal junction and weakening the cohesion between epidermis and dermis [1]. Protocol Incubation of human plasmin with labeled casein and EPERULINE PW (soybean trypsin inhibitor (SBTI) used as positive control) Incubation for 3 minutes at room temperature Measurement of the rate of hydrolysed casein by recording the increase of fluorescence intensity (excitation at 485 nm and emission at 538 nm) Fig. 8 Schema of protocol. Results Plasmin activity in % / control Control [SBTI] (%) Eperuline TM PW (%) 7% inhibition Mean ± SD n=2 Student s t test / control : p<.1 Fig. 9 Inhibition of human plasmin. Conclusion EPERULINE PW dose-dependently inhibited human plasmin activity with an IC5 (Inhibitory Concentration 5%) of.2%.

7 Inhibition of release of pro-inflammatory neuropeptide CGRP by sensory neurons (in vitro test) Aim To evaluate on sensory neurons the activity of EPERULINE PW against the release of the proinflammatory neuropeptide CGRP. Background Fig. 1 Culture of sensory neurons (labeled with antiβ-tubulin antibody, nuclei stained with Hoechst solution). The skin includes many nerve fibers, in particular the C fibers 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 proinflammatory 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, TNFa, PGE2), inducing a vasodilatation and an inflammatory process, which in turn potentiates the neuronal response [7, 11]. Cultured sensory neurons can be stimulated by membrane depolarization using KCl, or by the TRPV1 agonist capsaicin, leading to an increase of CGRP released in the culture medium. Protocol Culture of sensory neurons: differentiation by incubation for 1 days Treatment: Control EPERULINE PW (*) Incubation for 2 minutes at 37 C Stimulation of neurons: exchange of medium for medium with: Control KCl KCl + EPERULINE PW Capsaicin Capsaicin + EPERULINE PW Incubation for minutes Measurement of the rate of released CGRP Fig. 11 Schema of protocol. (*) Lack of cytotoxicity of EPERULINE PW was preliminary tested by a MTT test. Results 1 1 Rate of released CGRP in % / KCl 5 41 Control KCl at 4 mm KCl + Eperuline TM PW (%) % inhibition Rate of released CGRP in % / capsaicin 5 29 * 62 * 4 6% inhibition Control Capsaicin at 1 µm Capsaicin + Eperuline TM PW (%) n=6 Student's t test / KCl or capsaicin : p<.1 *: p<.1 Fig. 12 Inhibition of release of pro-inflammatory CGRP on KCl- and capsaicin-stimulated sensory neurons. Conclusion EPERULINE PW displayed a dose-dependent inhibition of released CGRP by cultured sensory neurons.

8 Anti-oxidative efficacy (in vitro tests) Aim To evaluate the activity of EPERULINE PW on lipoxygenase-catalyzed release of superoxide anions, and on UVA-induced oxidation of cell membrane lipids on human fibroblasts. Background The age-related micro-inflammation induces the release of reactive oxygen species (ROS), and results in the development of a chronic vicious cycle that leads to increasing skin damage. In the inflammatory process, lipoxygenases in the epidermis play a crucial role through the generation of pro-inflammatory leukotriens and superoxide anions. Moreover leukotriens cause the invasion of inflammatory cells like leucocytes in human skin thus prolonging the inflammatory process [12]. Furthermore, environmental stress such as UV or pollutants also generate ROS which provokes damage on all cellular and tissue constituents (lipids, proteins, sugars and nucleic bases) of human skin. UVA rays penetrate up to the dermis where they induce oxidative stress, mainly by activation of biological components which catalyze the formation of ROS like anion superoxide, hydrogen peroxide and singlet oxygen, and then lipoperoxidation of the cell membrane [13]. The lipoperoxides formed after UVA irradiation are decayed into malondialdehyde (MDA) which can cross-link with and alter many biological molecules like proteins and DNA, resulting in the aging of cells and of the extracellular matrix. Thus, oxidative stress was evaluated in vitro by measuring the level of released MDA. Protocol Culture of human fibroblasts in growth medium Incubation for 3 days at 37 C, CO 2 Incubation of linoleic acid and luminol in balanced solution Injection of EPERULINE PW and lipoxygenase (caffeic acid used as positive control) Measurement of the superoxide anions release by recording the luminescence on a period of 6 seconds Fig. 13 Schema of protocol: lipoxygenase-catalyzed release of superoxide anions. Exchange of growth medium by medium containing EPERULINE PW (vitamin E used as positive control) Incubation for 2 days at 37 C, CO 2 Exchange medium containing EPERULINE PW by a balanced salt solution, then UVA irradiation (2 J/cm²) Quantification of released MDA (*) Fig. 14 Schema of protocol: UVA-induced oxidation of cell membrane lipids on human fibroblasts. (*) Lack of cytotoxicity of EPERULINE PW was determined by measuring the level of cell proteins (Bradford's method). Results Rate of superoxide anions in % / control 1 5 * Control 4 Caffeic acid at.2% Fig. 15 Inhibition of release of superoxide anions catalyzed by lipoxygenase. * 66 * 38 * 9 Eperuline TM PW at:.1%.3%.1% 91% inhibition n=4 Student's t test / control (*) p<.1 Rate of MDA in % / UVA UV + vitamin E 6 Control UV at UV + Eperuline TM PW at: without UV 2 J/cm 2 at.3%.3%.1% n=9-12 Student's t test / UVA () p<.1 Fig. 16 Inhibition of UVA-induced oxidation of cell membrane lipids on human fibroblasts Conclusion EPERULINE PW displayed a dose-dependent inhibition of the lipoxygenase-catalyzed release of superoxide anions, with an IC5 of.21%. This anti-oxidative efficacy was further assessed on human fibroblasts by the dosedependent inhibition of MDA release induced by UVA oxidation, without any negative effect on cell viability % inhibition

9 Improvement of skin biomechanical properties and barrier function (clinical test) Aim To evaluate in vivo the efficacy of an emulsion containing 1% EPERULINE PW on the skin biomechanical properties and on the skin barrier function. Background Deformation (mm) Uf Ue Time (s) Fig. 17 Curve of extensiometry. Ur Ua Ur/Ue: elastic deformation rate, net elasticity Ur/Uf: elastic recovery rate Ua/Uf: ratio of total deformation, gross elasticity The chronic micro-inflammation induces molecular damage 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. The effect on biomechanical properties can be evaluated using Cutometer [14] measurements of the elastic deformation (Ur/Ue), the elastic recovery (Ur/Uf), and total deformation ratios (Ua/Uf), which have been shown to be negatively correlated with age [15] and severity of sagging [16]. The effect on the reinforcement of skin barrier function can be evaluated using Aquaflux by the measurement of Trans-Epidermal Water Loss (TEWL) [17]. Protocol 52 female volunteers, 3-65 years old Bi-daily randomized application on the face for 28 days Placebo emulsion Emulsion containing 1% EPERULINE PW Before (D) and after 28 days (D28) of treatment Measurement of the biomechanical properties of the skin on the temples by Cutometer SEM 5 (Courage & Khazaka) Fig. 18 Protocol of the clinical test. Measurement of the TEWL on the upper cheeks by Aquaflux AF (Biox Systems Ltd) Results 15 Percentage D28 / D 1 5 NS * Elastic deformation (Ur/Ue) 12% improvement / D NS * Elastic recovery (Ur/Uf) 1% improvement / D NS Total deformation (Ua/Uf) 1% improvement / D Placebo emulsion Emulsion containing Eperua falcata extract at 1% n=52 Paired Student's t test / D NS: not significant *: p<.5 : p<.1 Fig. 19 Improvement of skin biomechanical properties. Percentage D28 / D -5 NS 1% improvement / D Placebo emulsion Emulsion containing Eperuline TM PW at 1% -1 n=52 Paired Student's t test / D NS: not significant : p<.1-15 Fig. 2 Improvement of skin barrier function: reduction of TEWL. The tested products were well tolerated without any intolerance reactions observed during 28 days of application. The 28 days treatment with the emulsion containing EPERULINE PW significantly improved both the elastic deformation (Ur/Ue), the elastic recovery (Ur/Uf), and total deformation ratio (Ua/Uf) (p value =.131;.29 and.68 respectively for Ur/Ue, Ur/Uf and Ua/Uf). The increase of these three ratio parameters was between 1 and 12% comparatively to before treatment, whereas the placebo emulsion displayed only non significant 1-4% modification of these ratios (fig. 19). Comparison between placebo and EPERULINE PW treated sites is very close to significance with a p value of.58 and.84 for Ur/Ue and Ur/Uf respectively. The treatment of the skin with tested emulsions has reduced the TEWL (fig. 2). While this improvement was not significant for placebo treatment, it was significant (p=.64) for the emulsion containing EPERULINE PW and reached 1% comparatively to before treatment. Conclusion After 28 days of treatment, the cream containing 1% EPERULINE PW has shown significant restorative effects against agerelated degradation of skin biomechanical properties, loss of elasticity, firmness and tonicity involved in skin sagging. In addition, the EPERULINE PW 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.

10 General conclusion Chronic asymptomatic micro-inflammation appears strongly linked to the cutaneous aging process. Our results show that EPERULINE PW is able to inhibit in vitro the vicious cycle induced by this chronic low grade upregulation of the inflammatory response. It also is shown to lead in vivo to an improvement of the age-related loss of elasticity, firmness and tonicity involved in skin sagging, and reinforcing the integrity of the skin barrier function, and minimizing these visible signs of skin aging. Bibliography 1. Franceschi C et al.: Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 98, , 2 2. Bruunsgaard H et al.: Age-related inflammatory cytokines and disease. Immunol Allergy Clin North Am 23, 15-39, Cevenini E et al.: Systems biology and longevity: an emerging approach to identify innovative anti-aging targets and strategies. Curr Pharm Des 16, 82-13, Chung HY et al.: Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev 8, 18-3, Salminen A et al.: Genetics vs. entropy: longevity factors suppress the NF-kB driven entropic ageing process. Ageing Res Rev 9, , 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 3, , Peters EMJ et al.: Neuropeptide control mechanism in cutaneous biology: physiological and clinical significance. J Invest Dermatol 126, , Bechtel MJ et al.: Upregulation of cell-surface-associated plasminogen activation in cultured keratinocytes by interleukin-1b and tumor necrosis factor-a. Exp Cell Res 223, , Voegeli R et al.: Increased stratum corneum serine protease activity in acute eczematous atopic skin. Br J Dermatol 161, 7-7, Ogura Y et al.: Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction. Br J Dermatol 159, 49-6, Rossi R et al.: Cutaneous innervation and the role of neuronal peptides in cutaneous inflammation: a review. Eur J Dermatol 8(5), , Luo M et al.: Leukotriene synthesis by epithelial cells. Histol Histopathol , Dalle Carbonare et al.: Skin photosensitizing agents and the role of reactive oxygen species in photoaging J Photochem Photobiol B 14(1-2), , Barel AO et al.: Suction method for measurement of skin mechanical properties: the cutometer. In: Non-Invasive Methods & Skin. SERUP J ed, JEMEC GBE ed , Krueger N et al.: Age-related changes in skin mechanical properties: a quantitative evaluation of 12 female subjects Skin Res Technol 17, 141-8, Ezure T et al.: Influence of subcutaneous adipose tissue mass on dermal elasticity and sagging severity in lower cheek. Skin Res Technol 16, 332-8, Imhof RE et al.: New instrument for measuring water vapour flux density from arbitrary surfaces. IFSCC Magazine, 5, , 22

11 REFERENCE Eperuline TM PW LS 9627 DESCRIPTION Bark extract from Eperua Falcata DOSE OF USE 1% Regulatory data INCI (EU - US) Maltodextrin (and) Eperua Falcata Bark Extract CAS , EINECS , Preservative None Natural label Raw material conform to Ecocert standards of Natural and Organic cosmetics. Preliminary specifications Organoleptic characteristics rosy beige powder with a weak odor Ultra violet spectrum conform Odor evaluation versus standard corresponds to the standard Visual appaearance versus standard corresponds to the standard T.L.C Flavonoids conform Total sugars 9 - % Solubility at 1% in water soluble ph value (product at 1%) Microbiological control Upon request Formulation Stability Soluble in water, insoluble in oils and fats. Method of incorporation disperse EPERULINE PW in water at 1:3 (w/w) and introduce this solution during the finishing process below 4 C or at room temperature for cold processing. Suitable ph between 4 and 7 Optimal ph between 4 and 5.5 Patent APPLICATION Patents in force in EP (DE, FR, GB, IT, ES) (EP B1), US and KR, pending in JP STORAGE In its original packaging, at 15 - C SHELF LIFE 24 months MANUFACTURER BASF Beauty Care Solutions France SAS 3 rue de Seichamps Pulnoy (France)

12 EUROPE BASF Beauty Creations 49, avenue Georges Pompidou 993 Levallois-Perret Cedex FRANCE Tel: +33 () Fax: +33 () AMERICAS BASF Corporation 361 Sheep Pasture Road East Setauket, NY USA Tel: +1 (631) JAPAN & ASIA-PACIFIC BASF Japan Ltd. 21F Roppongi Hills Mori Tower, Roppongi, Minato-ku, Tokyo, JAPAN Tel: +81 () Fax: +81 () Edition may, 212 Although all statements and information in this publication are believed to be accurate and reliable, they are presented gratis and for guidance only, and risks and liability for results obtained by use of the products or application of the suggestions described are assumed by the user. SELLER MAKES NO WARRANTY OF ANY KIND, EITHER EXPRESS OR IMPLIED, BY FACT OR LAW, INCLUDING WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Statements or suggestions concerning possible use of the products are made without representation or warranty that any such use is free of patent infringement and are not recommendations to infringe any patent. The user should not assume that toxicity data and safety measures are indicated or that other measures may not be required. The claims and supporting data provided in this publication have not been evaluated for compliance with any jurisdiction s regulatory requirements and the results reported may not be generally true under other conditions or in other matrices. Users must evaluate what claims and information are appropriate and comply with a jurisdiction s regulatory requirements. Recipient of this publication agrees to (i) indemnify and hold harmless each entity of the BASF organization for any and all regulatory action arising from recipient s use of any claims or information in this publication, including, but not limited to, use in advertising and finished product label claims, and (ii) not present this publication as evidence of finished product claim substantiation to any regulatory authority.