126 SKIN BRIGHTENING membrane.7 Therefore, it degrades and removes
any already formed lipofuscin (Figure 1). Regarding both proteasome and autophagy,
it is known that their activity correlates with the lifespan of an individual organism.8
On the
other hand, it is also known that the activity of proteasome and autophagy decreases with age. This leads to a delayed degradation of proteins that have completed their roles in aged cells and the accumulation of oxidized, structurally denatured, and aggregated proteins.9 Based on these facts, naturally derived
ingredients with proteasome and/or autophagy activating properties are expected to have the ability to improve the clarity of the skin by reducing lipofuscin accumulation and brightening the skin. We believe that the prevention of lipofuscin formation is the most important step and have searched for naturally derived ingredients capable of activating proteasome.
Search for ingredients of natural origin that activate proteasome We screened approximately 400 types of plant-derived ingredients using activity of proteasome as an index. During the screening process, we used HEK293T cells derived from human embryonic kidneys, which are often used in cell culture tests. When proteasome activity in a steady
state is set to 100%, eight types of extracts showed activity of 150% or more. Among the hit ingredients, we selected juniper berry extract after consideration using no significant influence of differences in the production areas as well as stable activity for at least one year after the preparation of extract as indices. Juniper berry is the fruit of juniper
(Juniperus communis) which is a coniferous tree in the genus Juniperus of the cypress family that grows in the cold regions of North America, Europe, and Asia. It is a type of medical herb used since ancient times in essential oil, aromatic oil, and herb tea for its detoxification and wound healing effects. However, the proteasome activation effect of juniper berry extract was not known.
1.0% ■ 0.5% ■ Control ■
300 250 200 150 100
200 *** *** 150 *** 2h ** 4h *** *** 100 8h 24h Time after sample addition 2h 4h 8h 24h Time after sample addition *** *** *** ** *** 100 2h 4h 8h 24h Time after sample addition
Figure 2: Juniper berry extract's effect to activate proteasome and reduce lipofuscin accumulation. Concentration-dependent and time-dependent proteasome activation effect of juniper berry extract on HEK293T cells (left), human epidermal keratinocytes derived from adults (centre), and human epidermal keratinocytes derived from newborn (right) are shown assuming the steady state being 100% (n=4, mean±SEM, ***p<0.001, **p<0.01 vs. control by Dunnett's test in each time point). Figures in the lower section show the results of incubating human epidermal keratinocytes with or without juniper berry extract for a certain period and detecting lipofuscins in the cells using the Fontana-Masson staining method
PERSONAL CARE April 2024
www.personalcaremagazine.com *** *** *** 150 *** *** Juniper berry extract (0.5%) *** *** *** 200
Figure 1: Relationship among skin lightness, lipofuscin, proteasome, and autophagy
Juniper berry extract's effect to activate proteasome and reduce lipofuscin accumulation Figure 2 shows the results of the evaluation of juniper berry extract's effect to activate proteasome in HEK293T cells, human epidermal keratinocytes derived from adults, as well as human epidermal keratinocytes derived from newborns. When juniper berry extract was added to
HEK293T cells with a final concentration of 1%, proteasome activity increased to 200% or more after two hours. When it was added to human epidermal keratinocytes derived from adults or newborns with the final concentration of 1%, proteasome activity also increased to nearly 200% within two hours. This effect continued for approximately four
hours after addition and the state returned to an almost steady state 24 hours later. As the proteasome activation effect appears in a short time, within two hours, we believe that juniper berry extract makes proteasome hyperactive, rather than promoting the expression of proteasome itself. Then, we evaluated how the activation of proteasome induced by juniper berry extract
reduces the lipofuscin accumulation in cells. Human epidermal keratinocytes derived from newborns were incubated with or without juniper berry extract (0.5%) for a certain period. Then, accumulated lipofuscins in the cells
were stained using the Fontana-Masson staining method. As shown in Figure 2, a large number of brown dots derived from accumulated lipofuscin were seen in epidermal keratinocytes cultured without juniper berry extract (control group). Meanwhile, the number of brown dots
apparently reduced in epidermal cells cultured with juniper berry extract. The result suggests that adding juniper berry extract reduces lipofuscin accumulation in cells.
Identification of active components of juniper berry extract We tried to identify active components of juniper berry extract in order to reveal some of its action mechanism and found that the major active components responsible for the extract's effect to activate proteasome are anthricin and yatein which are lignans. Although lignans are characteristic
components found in Cupressaceae, it was not known that anthricin and yatein have an
Control
Proteasome activity (%)
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