The global beauty trend is shifting from “skin whitening,” which depends on one’s natural skin color, to “skin brightening,” which is a borderless concept. “Whiteness” is defined as skin color based on the amount of melanin, the black pigment produced by melanocytes. Although acquired factors also play a role, genetics have a strong influence on the whiteness. On the other hand, “bright skin,” which is often interpreted in terms such as “clarity,” depends on various acquired factors instead of melanin. 

One factor is lipofuscin, an aggregate of denatured proteins and lipid peroxides that accumulates within cells as the body ages. Lipofuscin decreases skin clarity by blocking light transmission, causing dullness. In response, we sought out juniper berry extract (JBE) from approximately 400 types of medical herbs as an ingredient of natural origin. It activates proteasome, an intracellular cleaning system that reduces lipofuscin accumulation. We developed it under the product name JuniperBright.¹ This article introduces JBE’s effect to activate proteasome, reduce lipofuscin accumulation, activate autophagy (another intracellular cleaning system) found through the identification of active ingredients as well as its beauty effects on human skin.

Skin Clarity and Light Relationship

One element that contributes to the clarity of skin is the “proper reflection of light.” When light hits the skin, some of it is reflected from the surface due to moisture and oil on the surface, but most of it penetrates the epidermal layer, the outer layer of the skin, as incident light and enters the skin. The incident light is absorbed and scattered inside the skin. It passes back through the epidermal layer again and is emitted as internally reflected light. 

The combined surface and internally reflected light from the skin appears to the observer as the brightness of the skin. During this stage, the more internally reflected light, the brighter the skin appears to the observer.² However, skin brightness has been reported to decrease with age.³ We focused on lipofuscin, an aggregate of denatured proteins and lipid peroxides that accumulate in cells as the body ages, as a factor that reduces light transmission in cells (Fig. 1).

Lipofuscin Interferes with Light

Lipofuscin, composed of “lipo” meaning lipid and “fuscus” meaning dark, causes yellow-brown spots that appear on aged skin.⁴ Lipofuscin does not only absorb light at a wide range of wavelengths,⁵ but also reduce light transmission because of its aggregation (Fig. 1). Reduction of lipofuscin accumulation in the cells of the epidermal layer is expected to increase the internal reflected light from the skin as well as decrease age spots, leading to a younger, and more beautiful skin.

To reduce lipofuscin accumulation requires the prevention of lipofuscin formation and the removal of formed lipofuscin. Proteasomes are complex proteases that selectively degrade proteins which have reached the end of their intracellular life cycle or have become dysfunctional due to oxidation or other structural changes.⁶ Rapid removal of denatured proteins generated within the cell prevents the formation of lipofuscin (Fig. 1). 

Autophagy is a major intracellular cleaning system, along with proteasome, that degrades cellular organelles such as aggregates and mitochondria, which are difficult for proteasome to break down, by internalizing them into the membrane.⁷ It degrades and removes any already formed lipofuscin (Fig. 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 the prevention of lipofuscin formation is the most important step and have searched for naturally derived ingredients capable of activating proteasome.

Juniper Berry Extract

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. We selected JBE 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 has been 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 JBE was unknown.

Activate Proteasome and Reduce Lipofuscin

Figure 2A shows the results of the evaluation of JBE’s effect to activate proteasome in HEK293T cells, human epidermal keratinocytes derived from adults, as well as human epidermal keratinocytes derived from newborns. When 1% JBE was added to HEK293T cells, 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 1.0%, proteasome activity increased 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 JBE makes proteasome hyperactive, rather than promoting the expression of proteasome itself.

Next, we evaluated how the activation of proteasome induced by JBE reduces the lipofuscin accumulation in cells. Human epidermal keratinocytes derived from newborns were incubated with or without JBE (0.5%) for a certain period. Then, accumulated lipofuscins in the cells were stained using the Fontana-Masson staining method. As shown in Figure 2B, a large number of brown dots derived from accumulated lipofuscin were seen in epidermal keratinocytes cultured without JBE (control group). Meanwhile, the number of brown dots were apparently reduced in epidermal cells cultured with JBE. Results suggest adding JBE reduces lipofuscin accumulation in cells.

Mechanism of Action

We tried to identify active components of JBE 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 (Figure 2C). Although lignans are characteristic components found in Cupressaceae, it was not known that anthricin and yatein activate proteasome. 

We discovered that, when JBE’s effect to activate proteasome is set to 100%, anthricin is responsible for approximately 90% and yatein is responsible for the remaining 10% as shown in Figure 2D. Furthermore, we demonstrates that anthricin binds to the proteasome and activates its protease activity but does not induce gene expression of the proteasome. Additionally, molecular dynamics simulations predicted its binding pockets and suggested the mechanism to enhance proteasome activity¹⁰ Finding new compounds that activate proteasome and its binding sites will contribute to the clarification of the molecular mechanism that controls proteasome activity.

JBE Activates Autophagy 

Successful identification of active components led to the discovery of a new effect of JBE. It has been reported that the anthricin analog causes autophagy activation.¹¹ We, therefore, evaluated the autophagy activation effect of JBE, anthricin, and yatein. As a result, as shown in Figure 3, all significantly activate autophagy during the period 8 to 24 hours. These results suggest that JBE also has an effect to activate autophagy. The extract not only prevents lipofuscin accumulation but also degrades it due to these functions.

Fewer Age Spots

A test was conducted to evaluate whether JBE’s effects to activate proteasome as well as autophagy and to reduce lipofuscin accumulation led to the improvement of lightness and reduction of age spots in human skin. An emulsion containing 1% JBE, or a placebo emulsion was applied to the half of the face of eight healthy men and women twice a day for four weeks. Internal reflected light was measured using TransluDerm. The L*a*b* value in the cheek area was measured using a color chromatometer, and the number of age spots in the cheek area was measured using Visia (Fig. 4).

In a TransluDerm test, white LED comes into contact with the skin and internal reflected light is detected using multiple detectors located in a straight line from the light source. The more the internal reflected light, the farther from the source light is detected. In the JBE group, the distance from the light source of detectors that reacted to the internal reflected light was significantly larger at week 4 than week 0. This result indicates that application of JBE creates a skin condition with more internally reflected light.

Next, skin lightness L-value, skin redness a-value, and yellowness b-value were measured using a color chromatometer. Significant improvement of L-value was observed after the test in the JBE group. There was no significant difference in a-value before and after the test in the JBE group or the placebo group (data not shown). Meanwhile, significant decrease of b-value was observed after the test in both JBE group and placebo group. There was a tendency that b-value reduced even more in the JBE group than in the placebo group. This result indicates that application of JBE leaves skin lighter and less yellowish.

Lipofuscin accumulation is a known cause of age spots that appear on aged skin. We evaluated the number of surface spots in the cheek area using Visia. In the JBE group, the number of age spots in the cheek area significantly reduced at Week 4 compared to Week 0. The result indicates that application of JBE not only lightens the skin tone but also decreases age spots on the skin.

Conclusion

This article introduces JBE as a new ingredient of natural origin that activates proteasome and autophagy serving as cleaning systems in cells, reduces lipofuscin, and leaves the skin bright and clear with less age spots. For skin clarity involves, not only lipofuscin but various acquired factors such as glycated products (AGEs), gaps between epidermal cells, slight unevenness on skin surface, shape of pores and moisture content. Combination of care for these factors is expected to achieve borderless brightness that does not depend on natural skin color. We would be delighted if “JuniperBright,” the JBE we developed, contributes to the development of products that help to achieve beautiful skin.

About the Author
Kotaro Sakamoto is senior principal scientist at Ichimaru Pharcos Co., Ltd. 
sakamoto-kotaro@ichimaru.co.jp

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