Skin Hyperpigmentation: Understanding the Influence of the Exposome

24.05.2022

SymBright^® 2036 & SymBright^® PLUS

An individual’s totality of environmental exposure was defined (by C.P. Wild) in 2005 as ‘exposome’. Since then the definition has been adapted to the skin exposome including inter alia sun radiation, pollution, tobacco, temperature and stress as external factors resulting in skin aging. In our research program the effect of selected factors of the exposome on skin pigmentation was explored with the aim to develop an active to protect the skin against the exposome-induced dark spot formation, one of the aging processes of skin. As external stressors UV irradiation, visible light irradiation and diesel exhaust particles were used. The active molecule developed within the project is SymBright 2036® (INCI: sclareolide). Sclareolide is a sesquiterpene lactone obtainable from clary sage (Salvia sclarea L.).

Introduction

Stress, pollution, UV rays, but also visible light, infrared rays… the list of external elements agressing the skin keeps getting longer. These all seem to trigger a common reaction sequence in the skin involving the induction of inflammation and the production of melanin. If this sequence is fully part of the skin natural defense system, the later is often over-reacting provoking pigmentation disorders and darks spots. Skin lightening has come a long way from the basic ‘whitening’ approach, aimed at ethnic consumers with darker skin to new targets like illuminating skin and spot-targeting to reduce the appearance of brown / age spots. For many consumers uneven skin tone can be a source of shame. 58% of consumers feel bad with an uneven skin tone [1]. The demand for skin perfection is rising in women of all ages. For these purposes it is important to understand the pigmentation mechanisms in the epidermis. It is well accepted that tyrosinase inhibitors in cosmetics contribute to the reduction of the pigmentation of the skin. But to protect against the formation of freckles and melasma the cross-talk of keratinocytes and melanocytes has to be taken into account. The stimuli resulting in this undesirable inhomogeneous pigmentation are divers: UV irradiation is a well-known pigmentation enhancer; acne scars and depilation or shaving lesions are increasingly demanding effective products [2]; but also air pollution was identified recently as a major inducer of lentigines [3,4]. All stimuli unify the underlying mechanism of keratinocytes releasing a bunch of cytokines triggering melanocytes to proliferate, differentiate, produce melanin and transfer melanosomes to keratinocytes [5], with the most prevalent mechanism known as postinflammatory hyperpigmentation (PIH). As pigment spots may only resolve over a protracted period of time if left untreated [6] a protection against PIH combined with a depigmenting effect is the most promising cosmetic treatment. In recent years the view on pigmentation induced by sunlight was diversified from the only UV part to the additional contribution of visible light. As approximately 52% of the sun contributes to visible light at sea level [7] and this part of the sun is penetrating deep into the skin the interest in the effect of visible light on the skin physiology was increasing in recent years [8]. One important observation was a tanning of the skin despite the use of UV protection products leading to the need of a deeper look into the parts of the sunlight beyond UV linked to pigmentation. Duteil et al. [9] nicely showed that the pigmentation induced by the blue/violet part of visible light is more persistent compared to the UV induced pigmentation. The interest in products protecting against this persistent hyperpigmentation is increasing. In vitro models to investigate the mechanisms and identify potent actives are under development.

Materials and Methods

Skin pigmentation in vitro: B16V melanoma cells were seeded into a 96-well microtitre plate. After adherence, various concentrations of the test substances and 10 nM α-MSH added. After 96h incubation, alkaline extraction of the cellular melanin was performed, the absorption was measured at 400 nm. Skin pigmentation ex vivo: Full thickness human skin explants (7 x 3 mm; Ø x thickness) obtained from abdominal plastic surgery were transferred into culture plates and placed on a cotton pad, soaked with culture medium. 4-6 skin explants were used per test concentration and placebo. SymBright 2036^® formulated into a hydrodispersion gel was topically applied once per day. On day 6, histological sections were prepared and melanin was quantified by image analysis after Fontana-Masson staining. Kojic acid served as positive control and was applied at a concentration of 0.1% in DMSO. Visible light-induced hyperpigmentation in vitro: Human epidermal melanoma cells A375 were cultured in 6-well plates. The cells were exposed to visible light (480 J/cm2) with the Hydrosun 750 equipped with KG1 filter (to filter out infrared A radiation) and HBM1 (Hydrosun Medizintechnik GmbH, Müllheim, Germany). 48 h prior to and past to the irradiation the cells were treated with test compounds in non-cytotoxic concentrations. Melanocytes were lysed in a solution of NaOH. Melanin was quantified by measurement of absorbance at 405 nm and calculation based on a melanin standard curve. Visible light-induced hyperpigmentation ex vivo: Finished formulations were applied on ex vivo human skin explants from abdominal surgery of a donor with phototype IV (Fitzpatrick scale). 48 h after the formulations were removed with a cotton pad. Skin explants were exposed to visible light (480 J/cm2) with Hydrosun 750 equipped with KG1 filter. Formulations were reapplied afterwards. 48 h later skin sections were prepared and melanin was stained by Fontana-Masson. Analysis of melanin was done semi-quantitatively by image analysis.

Pollution-induced hyperpigmentation

Model DEPs: Standard reference materials (SRMs) SRM1650b (mean particle diameter 0.18 μm ) of the National Institute of Standards and Technology, Gaithersburg, MD, USA, were used as appropriate surrogates for authentic street particulate matter. The SRMs were suspended in phosphate buffered saline and were sonicated for 1 min and then directly added to the keratinocytes at a concentration of 1.5 μg/cm2. RNA Isolation and PCR: Total RNA was prepared as described earlier [10]. Cyp1A1 and MMP-1 gene were quantified 6 h after application of DEPs, IL-6 and POMC gene were quantified 24 h after application of DEPs by qRT-PCR. Three samples for each condition have been processed with 2 determinations each.

Clinical study

24 Asian adult subjects with Phototype II to III applied twice daily from Day 0 to Day 48 on their forearm a placebo formulation and the same formulation with 0.2% SymBright 2036^®. In order to induce pigmentation they were exposed to a progressive dose of UV (0,7MED to 1MED) on D0, D1, D2 and D3. The maximum pigmentation was achieved on D7. Therefore the pigmentation variation is normalized to D7.

Results

SymBright 2036® showed a potent lightening effect when tested on B16V melanoma cells in vitro with an IC50 of 10.2 μM (Fig. 1A). In the next step, SymBright 2036^® was tested on ex vivo human skin (photo-type intermediate) dosed at 0.1% in a hydrodispersion gel formulation. A significant lightening effect was observed with a reduction of 41% versus placebo (Fig. 1B). In comparison the positive control kojic acid applied at 0.1% in DMSO reduced melanin content by 30%.

[caption id="attachment_139780" align="aligncenter" width="557"]

Figure 1: Inhibition of melanogenesis: SymBright 2036^® inhibited melanin production in B16V cells in vitro (A). Melanin in human ex vivo skin explants was reduced by 0.1% SymBright 2036^® applied in a gel formulation after 6 days; positive control kojic acid was applied 0.1% in DMSO (B).[/caption] Due to in vitro experiments on B16V cells as well as RTq-PCR of ex vivo explants it was shown that the lightening activity is based on a direct effect on melanocytes but also an altered crosstalk between keratinocytes and melanocytes including POMC as one of the relevant down-regulated genes. The protection against visible light-induced hyperpigmentation was demonstrated in vitro on human A375 cellline with 0.00079% active inhibiting significantly 70% of the induction (data not shown). [caption id="attachment_139782" align="aligncenter" width="481"]

Figure 2: Ex vivo inhibition of VIS-light induced-melanin synthesis.[/caption] [caption id="attachment_139783" align="aligncenter" width="500"]

Figure 3: Air-pollution induced stimulation of POCM gene expression : SRM1650b stimulates POMC gene expression significantly. SymBright 2036^® prevents this increase significantly.[/caption] Hyperpigmentation due to urban pollution was explored in vitro on HaCaT cells by applying diesel exhaust particles SRM1650b resulting in increased release of POMC protein. The active dosed 0.00025% was able to reduce this increase by 49%. In an in vivo study the active was applied at 0.2% to the forearm of Asian subjects. Hyperpigmentation was induced by UV irradiation on 4 successive days. The active was able to reduce the hyperpigmentation within 48 days significantly vs placebo. [caption id="attachment_139784" align="aligncenter" width="579"]

Figure 4: in vivo UV-induced pigmentation — 24 Asian adult subjects -Product Application from D0 to D48 - Exposure to 0,7MED to 1MED on D0, D1, D2 and D3 Maximum pigmentation achieved on D7. Therefore the pigmentation variation is normalized to D7. SymBright 2036^® shows a good tendency to prevent from UV-induced pigmentation with a significant result after 48 days of application.[/caption]

Conclusion

SymBright 2036^®, produced in high purity from clary sage is a natural, safe and sustainable product. Clary sage contains sclareol in stems, leaves and flowering parts. Biotransformation of sclareol results in sclareolide. By subsequent extraction and concentration processes sclareolide is obtained in high purity. As sclareolide is used since decades for fragrance production and in fragrance mixtures the safety profile is well-known and SymBright 2036® is a China registered cosmetic ingredient. SymBright 2036^® optimally protects the skin from overreactions towards harmful environmental influences which can lead to permanent hyperpigmentation disorders on face and body. It protects the skin against the harmful effects of air pollution and UV rays and blue light with visible results after just a few weeks of use. The skin’s natural brightness is maintained or even returns. A protection against hyperpigmentation, induced by UV or air pollution, respectively, combined with a depigmenting effect is the most promising cosmetic treatment against spots and inhomogeneous pigmentation. By SymBright 2036® these properties are combined in one active making it an attractive material for deodorant, depilation, and acne applications but also the day cream to protect against harming influences all day long providing illuminating skin and reducing the appearance of brown / age spots.

Bright Partner for Bright Skin

As a pioneer explorer of the exposome, Symrise is constantly developing new solutions for pigmentation. Dark spots are now perceived as the new wrinkles, and Symrise has been exploring the paths to tackle this specific concern and looking for a bright partner with optimized efficacy. To capitalize on our blockbuster SymBright^® 2036 – we looked for a smart & efficient combination. Niacinamide – vitamin B3 – appeared as a perfect match to enhance the efficacy of SymBright^® 2036 thanks to its multiple virtues leading to the development of SymBright^® PLUS, a ready-to-use liquid blend.

Testing Efficacy of Symbright^® PLUS

UV light can induce melanogenesis in the skin through two major distinct pathways: by direct activation of the melanocyte or via an indirect pathway by generating Reactive Oxygen Species (ROS) e.g. in surrounding keratinocytes. These keratinocytes react to ROS by producing stress signals which are then secreted and subsequently stimulate melanogenesis. [caption id="attachment_139785" align="aligncenter" width="624"]

Figure 5: Schematic representation of direct and indirect route for melanogenesis induction by UV light.[/caption] The newly launched ingredient SymBright^® PLUS has been developed as a skin brightener. Therefore, we evaluated its efficacy on human skin explants exposed to UV irradiation. As the generation of ROS as starting point for the indirect activation pathway is a very quick process, we studied the capability of SymBright^® PLUS to reduce the amount of ROS in skin explants irradiated with 60 J/cm2 UVA. In figure 6, cross sections of skin explants are displayed with a green fluorescence signal indicative of the presence of ROS in the tissue. [caption id="attachment_139786" align="aligncenter" width="979"]

Figure 6: Fluorescence staining (DCFH-DA) of histological cross sections from human skin explants (female donor, age 51, ITA angle 36° “intermediate”); intensity of fluorescence is correlated to the presence of ROS in the tissue. Scale bars represent 100 μm.[/caption] Quantification of the fluorescence allows the evaluation of the ROS reducing activity of the test samples. SymBright^® PLUS led to a 66% decrease in ROS score vs UV-irradiated control. Also tested were the components making up SymBright^® PLUS in their respective concentrations, and none of the single ingredients reached the efficacy in ROS reduction of SymBright^® PLUS. This result demonstrates the superiority of SymBright^® PLUS vs its single components in terms of ROS reduction in the human skin. In a second experiment, we analyzed the effect of SymBright^® PLUS on UV-induced pigmentation. Human skin explants (female donor, age 43, ITA angle 24° “tanned”) were exposed to UVB irradiation daily for 7 days with a dose of 75 mJ/cm2. Quantitative evaluation of melanin content in the skin explants was performed using Fontana-Masson staining. SymBright^® PLUS reduced melanin in the skin explants by 26% vs the UV control (see figure 7). [caption id="attachment_139787" align="aligncenter" width="799"]

Figure 7: Assessment of melanin content by Fontana Masson staining in human skin explants after 7d of treatment with the test items and subsequent exposure to 75 mJ/cm² of UV B irradiation. The black staining is indicative of the presence of melanin and was quantified by image analysis. Scale bars represent 100 μm.[/caption] Changes in melanin content can be caused by degradation of existing melanin or by reduction of the biosynthesis of the pigment. To assess the influence of SymBright^® PLUS on melanogenesis, we analyzed the abundance of Melan-A as marker of de novo formation and maturation of melanosomes, the organelles in which melanogenesis takes place. Treatment of UVB-irradiated skin explants with SymBright^® PLUS led to a 64% reduction of Melan-A signals compared to the UV-treated vehicle control. Also, SymBright^® PLUS outperformed its single compounds in reducing Melan-A signals, indicative of de novo melanogenesis. [caption id="attachment_139788" align="aligncenter" width="717"]

Figure 8: Representative image of Melan-A signals in cross sections of human skin explants after 7 d treatment with test items and daily irradiation with 75 mJ/cm2 UVB. Image shown is vehicle control with UVB irradiation. Cells colored in deep red (examples shown by black arrows) contain Melan-A and therefore are considered to be engaged in de novo melanogenesis. Scale bar represents 100 μm.[/caption] Taken together our findings indicate a very good reduction of UV-induced ROS species in human skin explants, as well as significant reduction in melanogenesis by SymBright^® PLUS, thus potentially exerting an effect both on the direct and the indirect pathway for UV-induced melanogenesis. The product outperforms its single components in terms of ROS inhibition and de novo melanogenesis, clearly demonstrating the added value of the product. Conclusion SymBright^® PLUS is a versatile and - in vivo proven - efficient solution for the prevention of uneven skin pigmentation and hyperpigmented spots. Thanks to its easy-to-use liquid form, it can be used in multiple applications such as everyday face & body care, anti-spot treatments, tissue masks and more. Referanslar / References: 1. CMI Veri Kaynağı: Symrise Kozmetik Bileşenleri Tüketici veri tabanı 2012-2013 (10 pazarda 36 000 tüketici) 2. Ortonne ve Bissett (2008) J Araştırması Dermatol semptom İşlem 13(1):10-4 3. Vierkötter ve diğerleri (2010) J Invest Dermatol 130(12): 2719-2726 4. Nakamura ve diğerleri (2015) Uzm . Dermatol 24(6): 407-11 5. Yamaguchi ve Hearing (2009) Biofactors 35(2): 193-9 6. Davis ve Callender (2010) J Clin Esthet Dermatol 3(7):20-31 7. CIE Teknik Raporu No 85,1989, tablo 4 'Deniz seviyesinde küresel güneş ışınımı' 8. Dupont, E., Gomez, J., Bilodeau , D., Int J Cosmet bilim _ 35(3 ): 224-32 (2013) 9. Duteil, L., Cardot -Leccia, N., Queille -Roussel, C., Maubert, Y., Harmelin , Y., Boukari , F., Ambrosetti , D., Lacour, JP, Passeron, T., Pigment Hücresi Melanom Res 27(5 ): 822-826 (2014) 10. Grether -Beck ve diğerleri (2008) Exp Dermatol 17 :771-79