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EYE AREA CARE Control ■ 3.0% HB Complex ■


120 100 80 60 40 20 0


40 35 30 25 20 15 10 5 0


Haeme Haeme + UV


Figure 3: HB Complex reduces melanin production in epidermal skin models containing melanocytes and stressed by haeme and UV radiation.


and pro-inflammatory. As keratinocytes have the ability to produce HO-1 and can be exposed to haeme, it is important to verify that the effect of haeme on HO-1 production by keratinocytes is not negatively influenced.


Method Human keratinocytes (HaCaT) were pretreated with HB Complex for 24 hours. Haeme (5 µM) was added for different periods of time. The determination of HO-1 was performed by ELISA (R&D Systems, Inc.; KCB3776). Relative fluorescence units were determined at 540 nm (ex.)/600 nm (em.). Non-treated cells were set at 100%.


Results Haeme treatment of keratinocyte (HaCaT) cells at first led to an increase of HO-1 production, but prolonged exposure (48 h) to haeme led to a clear reduction of HO-1 production (Fig 1). As haeme is proinflammatory and pro-oxidative, this is likely a result of the prolonged stress the cells are exposed to. HB Complex allows the cells


VOLUNTEER 1 : T=0 Normal osmolarity Hyperosmotic (+100mOs m/l)


Figure 4: HB Complex increases VEGF-C production in lymphatic endothelial cells.


to deal with the presence of haeme, leading to an increased production of HO-1 after prolonged exposure to haeme. This effect could not be observed in the control cells. The experiment mimicked the in vivo situation in dark circles, where prolonged haeme exposure can have deleterious effect on the skin cells. HB Complex showed that it can compensate effectively for these effects.


Autophagy Autophagy is a process by which cells can recycle old dysfunctional organelles and protein aggregates. The accumulation of such “cellular waste” is an important feature in cellular ageing. Induction of autophagy, therefore, is a sensible anti-ageing approach which increases cellular longevity and functionality. Autophagy is a process with which


keratinocytes degrade melanin-containing melanosomes. In this context autophagy is described as an important factor in regulating skin colour, and induction of autophagy is therefore an effective approach to lighten dark circles.


VOLUNTEER 1 : T=56


Method Human keratinocytes (HaCaT) were pre-treated with HB Complex for 24 hours. Cells were treated with 2 µM chloroquine (inhibition of autophagy flux). Cells were incubated for a further 48 hours w/wo HB Complex. Determination of LC3B was performed by ELISA using LC3B (D11) XP® mA (New England Biolabs, Inc.). The detection antibody was an anti-IgG (H+L), F (ab’)2 fragment (Alexa®488) (New England Biolabs, Inc.). The fluorescence was measured at 485 nm (ex.) and 535 nm (em.) in a fluorescence reader (Spectramax Paradigm, Molecular Devices, LLC.). Non-treated cells served as controls and were set at 100%.


Results HB Complex at both 0.5% and 1.0% strongly induced autophagy in keratinocytes (HaCaT) (Fig 2). This indicates that it strongly supports the cellular anti-ageing processes and can increase cellular functionality (as demonstrated above). HB Complex supports the cellular processes in breaking down melanosomes, leading to skin lightening activities.


Melanogenesis under the influence of haeme and UV radiation Pigmentation plays an important role in the appearance of dark circles. Haeme is proinflammatory and pro-oxidative. It is an inducer of melanogenesis. Like haeme, UV light induces inflammatory and oxidative processes in the skin. It is therefore of interest to study whether the treatment with haeme and haeme together with UV radiation has an influence on melanin synthesis, and if HB Complex can reduce melanin synthesis induced by haeme and UV light.


VOLUNTEER 2 : T=0 VOLUNTEER 2 : T=56


Method Week 1: Human epidermal equivalents with melanocytes (epiCS-M, CellSystems GmbH) were treated for one week with 10 µM haeme on the basolateral side. Some of the skin models were irradiated with 0.3 J/cm2


UVA + 0.03 J/cm2 Figure 5: 3% HB Complex clearly reduces the surface area of dark circles after 56 days of use. www.personalcaremagazine.com


UVB once daily. Week 2: Haeme treatment and irradiation were stopped, and the models were treated with either water (control) or 3% HB Complex (in water) for 7 days. Melanin content of the skin models was extracted and quantified by photometry. Non-treated skin models served as controls and were set at 0%.


March 2021 PERSONAL CARE Control ■ 0.5% HB Complex ■


55


Increase in melanin production (%)


Increase in VEGF-C production (%)


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