The Importance of 11α-OH, 15-oxo, and 16-en Moieties of 11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Its Inhibitory Activity on Melanogenesis

Cosmetic industries have an interest in exploring and developing materials that have the potential to regulate melanin synthesis in human skin. Although melanin protects the skin from ultraviolet irradiation, excess melanin can be undesirable, particularly on the face where spots or freckles are associated with an appearance of aging. In this study, we found that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA) in Pteris dispar Kunze strongly inhibited melanin synthesis by suppressing tyrosinase gene expression. The melanogenic transcription factor microphthalmia-associated transcription factor (MITF) is required for this suppression. However, 11α-OH KA did not modulate the expression level or activity of MITF. Structure-activity relationship analyses suggested that the 11α-OH, 15-oxo, and 16-en moieties of 11α-OH KA are essential for the suppression of melanin synthesis. On the other hand, the 19-COOH moiety is important for preventing cellular toxicity associated with 11α-OH KA and its related compounds. These results suggest that 11α-OH KA is an attractive target for potential use in the production of cosmetic items.



Melanin plays a crucial role in protecting the skin from ultraviolet (UV) components of sunlight. In addition, melanin neutralizes free radicals and scavenges toxic drugs and chemicals [1]. However, generation of excess melanin causes visible hyperpigmentation of the epidermis, resulting in freckles, melasma, and age spots [2].

Hyperpigmentation results from an increased number or activity of melanocytes [3]. Melanin synthesis is regulated by tyrosinase (EC, which catalyzes hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine and its subsequent oxidation to dopaquinone. Dopaquinone is converted to melanin via enzymatic and nonenzymatic reactions [4]. To achieve skin whitening, control of tyrosinase activity and its expression level is important. A number of substances, including linoleic acid [5], hinokitiol [6], kojic acid [7], hydroquinone [8], catechols [9], and salicylate [10], are widely used in cosmetic items where they act as tyrosinase inhibitors.

The regulation of tyrosinase expression levels in melanocytes is a multistep process. First, UV radiation stimulates keratinocytes to synthesize and secrete α-melanocyte-stimulating hormone (α-MSH) [11]. α-MSH then binds to the melanocortin 1 receptor on the melanocyte membrane, which initiates cAMP signaling in the melanocytes. cAMP signaling activates the transcription factor cAMP response element-binding protein (CREB), which induces the melanogenic transcription factor microphthalmia-associated transcription factor (MITF) [12,13]. In turn, MITF binds to the tyrosinase promoter and upregulates the expression of this gene. We have recently identified carnosol and canosic acid from Callicarpa longissima as candidates for skin whitening reagents that act by targeting MITF expression [14].

Here, we report that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA), an ingredient in the Pteris dispar Kunze leaf extract strongly inhibited melanin synthesis in B16F10 melanoma cells and in a three-dimensional (3D) human skin epidermal model. Although 11α-OH KA did not alter the expression level or activity of MITF in cAMP-agonist (forskolin, Fsk)-treated cells, it significantly suppressed tyrosinase expression levels. Structure-activity relationship analyses revealed that the 11α-OH, 15-oxy, and 16-en moieties were essential for melanogenesis inhibitory activity of 11α-OH KA.

Materials and Methods


Authentic 11α-OH KA and related kaurens were obtained from BioBioPha (Shanghai, China). Antibodies were obtained as follows: anti-tyrosinase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-MITF (Thermo Fisher Scientific Inc., Waltham, MA, USA), anti-GAPDH (WAKO, Kyoto, Japan), anti-CREB (Genscript, Piscataway, NJ, USA), and p38, pp38, ERK1/2, pERK1/2, and pCREB (Cell Signaling Technology, Danvers, MA, USA). Antigen-antibody interactions were visualized using Chemi Doc XRS (Bio-Rad, Hercules, CA, USA) with the Chemi-Lumi One Super reagent (Nacalai Co. Ltd., Kyoto, Japan) or using the chromogenic reagent immunostain horseradish peroxidase (ATTO Co., Ltd., Tokyo, Japan).

Purification and Analysis of 11α-OH KA

Fifty grams of dried powder made from the leaves of P. dispar Kunze was soaked in 500 mL of methanol, and the resulting extract was passed through 50 g of activated charcoal and was then concentrated to approximately 10 mL (forming viscous precipitates) by evaporation. Viscous precipitates were first washed with 10 mL chloroform and were then re-resolved with 10 mL of methanol to which 500 mL chloroform was added. After removal of unresolved materials by filtration, solvents were re-evaporated. Substances were then concentrated to approximately 20 mL and were applied onto a silica gel column (100 mL; Nacalai, Kyoto, Japan). Melanin-suppressing substances were eluted as 2 peaks when chloroform:methanol (85:15) was used as the eluate. The latter peak fraction was concentrated and applied onto a C18-ODS column (4.6 × 50 mm, Cosmosil MS-II; Nacalai). The main peak was collected, and substances were recovered in ethyl acetate and re-concentrated by evaporation. The precipitates were then dissolved in 1 mL of ethanol to be hydrolyzed with 9 mL of 1% H2SO4-water at 100°C for 30 min. The hydrolyzate was recovered in ethyl acetate, evaporated, suspended in 20% acetonitrile-water, and applied again onto the C18-ODS column. The main peak was collected, and substances were again recovered in ethyl acetate and re-concentrated by evaporation.

Fr. 6 and 6′ (hydrolyzed) were purified by recycling HPLC onto an ODS column (15 × 150 mm i.d., SunFire Prep C18 OBD, Waters; solvent system, 20% acetonitrile/water) to obtain 11α-OH KA (2.0 mg).


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