Nutr Res Pract.  2018 Feb;12(1):3-12. 10.4162/nrp.2018.12.1.3.

Sageretia thea fruit extracts rich in methyl linoleate and methyl linolenate downregulate melanogenesis via the Akt/GSK3β signaling pathway

Affiliations
  • 1Faculty of Biotechnology, College of Applied Life Sciences, SARI, Jeju National University, 102, Jejudaehak-ro, Jeju-si, Jeju 63243, Korea. somikim@jejunu.ac.kr
  • 2Subtropical Horticulture Research Institute, Jeju National University, Jeju 63243, Korea.
  • 3Subtropical Tropical Organism Gene Bank, Jeju National University, Jeju 63243, Korea.

Abstract

BACKGROUND/OBJECTIVES
Sageretia thea is traditionally used as a medicinal herb to treat various diseases, including skin disorders, in China and Korea. This study evaluated the inhibitory effect of Sageretia thea fruit on melanogenesis and its underlying mechanisms in B16F10 mouse melanoma cells. The active chemical compounds in anti-melanogenesis were determined in Sageretia thea.
MATERIALS/METHODS
Solvent fractions from the crude extract were investigated for anti-melanogenic activities. These activities and the mechanism of anti-melanogenesis in B16F10 cells were examined by determining melanin content and tyrosinase activity, and by performing western blotting.
RESULTS
The n-hexane fraction of Sageretia thea fruit (HFSF) exhibited significant anti-melanogenic activity among the various solvent fractions without reducing viability of B16F10 cells. The HFSF suppressed the expression of tyrosinase and tyrosinase-related protein 1 (TRP1). The reduction of microphthalmia-associated transcription factor (MITF) expression by the HFSF was mediated by the Akt/glycogen synthase kinase 3 beta (GSK3β) signaling pathway, which promotes the reduction of β-catenin. Treatment with the GSK3β inhibitor 6-bromoindirubin-3'-oxime (BIO) restored HFSF-induced inhibition of MITF expression. The HFSF bioactive constituents responsible for anti-melanogenic activity were identified by bioassay-guided fractionation and gas chromatography-mass spectrometry analysis as methyl linoleate and methyl linolenate.
CONCLUSIONS
These results indicate that HFSF and its constituents, methyl linoleate and methyl linolenate, could be used as whitening agents in cosmetics and have potential for treating hyperpigmentation disorders in the clinic.

Keyword

Sageretia thea; melanins; methyl linoleate; microphthalmia-associated transcription factor

MeSH Terms

alpha-Linolenic Acid*
Animals
Bleaching Agents
Blotting, Western
Camellia*
China
Fruit*
Gas Chromatography-Mass Spectrometry
Hyperpigmentation
Korea
Linoleic Acid*
Melanins
Melanoma
Mice
Microphthalmia-Associated Transcription Factor
Monophenol Monooxygenase
Phosphotransferases
Plants, Medicinal
Skin
Bleaching Agents
Linoleic Acid
Melanins
Microphthalmia-Associated Transcription Factor
Monophenol Monooxygenase
Phosphotransferases
alpha-Linolenic Acid

Figure

  • Fig. 1 Effects of the n-hexane fraction of Sageretia thea fruit (HFSF) on cell viability and melanin content in B16F10 cells. (A) Melanin content was examined after treatments with 25 and 50 µg/mL of the Sageretia thea fruit crude extract and its fractions or 20 µM of resveratrol. The melanin levels visualized after treatment with the Sageretia thea fruit methanol extract and its fractions at concentration of 50 µg/mL. Cells stimulated with α-melanocyte stimulating hormone (α-MSH) for 48 h. (B) Cell toxicity of the HFSF was evaluated by MTT assay. (C) Melanin content was visualized and examined after treatments with the indicated concentrations of the HFSF or known whitening agents (AR, arbutin 2 mM; KA, kojic acid 400 µM; RSV, resveratrol 20 µM). Cells were stimulated with α-MSH for 48 h. Data are represented as means ± SD. *P < 0.05, **P < 0.01 versus α-MSH (500 nM) control; #P < 0.01 versus control. ME, methanol extract of Sageretia thea fruit; HF, n-hexane fraction from ME (HFSF); CF, chloroform fraction from ME; EF, ethyl acetate fraction from ME; BF, n-butanol fraction from ME; WF, water fraction from ME.

  • Fig. 2 The n-hexane fraction of Sageretia thea fruit (HFSF) suppresses tyrosinase activity and expression by decreasing MITF expression. B16F10 cells were treated with the HFSF or resveratrol (RSV, 20 µM) in medium containing α-MSH. (A) Intracellular tyrosinase activity and (B) in situ tyrosinase activity were determined after a 48 h treatment with the HFSF or resveratrol. Images were captured using microscopy Bar = 20 µm. (C) The expression of tyrosinase and tyrosinase-related protein 1 (TRP1) protein was detected by western blotting after the HFSF treatment for 48 h. (D) Protein expression levels, including MITF, Akt, p-Akt, GSK3β, p-GSK3β, and β-catenin were examined by western blotting. Cells were treated with the HFSF for 4 h. (E) The effects of the HFSF (100 µg/mL) on β-catenin expression were examined by western blot in the absence or presence of 6-bromoindirubin-3'-oxime (BIO, 2 µM) for 4 h. Data are represented as means ± SD.

  • Fig. 3 Effects of sub-fractions from the n-hexane fraction of Sageretia thea fruit (HFSF) on anti-melanogenesis in B16F10 cells. (A) Melanin content was examined after treatment with the HFSF, sub-fraction H-3 from the HFSF (H-3), sub-fraction H-3-1 from H-3 (H-3-1), or resveratrol (RSV, 20 µM) in medium containing α-MSH for 48 h. Melanin levels were visualized in the 50 µg/mL treatment concentration. Representative GC-MS chromatogram of HFSF (B), H-3 (C), and H-3-1 (D). Assigned peak numbers correspond to the number of identified compounds in Table 1. (1) methyl palmitate, (3) methyl linoleate, (4) methyl linolenate, (6) methyl stearate. Data are represented as means ± SD. *P < 0.05, **P < 0.01 versus α-MSH (500 nM) control.

  • Fig. 4 Effects of methyl linoleate (ML) and methyl linolenate (MLN) on melanogenesis in B16F10 cells. Cell toxicity of methyl linoleate (A) and methyl linolenate (B) was evaluated by the MTT assay. (C) Melanin content was visualized and determined after the ML, MLN, or positive whitening agent treatments (AR, arbutin 2 mM; KA, kojic acid 400 µM; RSV, resveratrol 20 µM) in α-MSH-supplemented medium for 48 h. Intracellular tyrosinase activity (D) and in situ tyrosinase activity (E) were determined after the ML, MLN, or resveratrol treatments in α-MSH-supplemented medium for 48 h. Images were captured using microscopy. Bar = 20 µm. Data are represented as means ± SD. **P < 0.01 versus α-MSH (500 nM) control; #P < 0.01 versus control.

  • Fig. 5 Effects of methyl linoleate (ML) and methyl linolenate (MLN) on melanogenic-related proteins in B16F10 cells. (A) Cells were treated with ML or MLN (50 µM, 100 µM) in the absence or presence of α-MSH (500 nM) for 48 h. Western blotting was conducted to examine the expression levels of tyrosinase and TRP1 protein. (B) Cells were treated with ML or MLN (100 µM) in the absence or presence of α-MSH for 4 h. The expression levels of protein including MITF, Akt, p-Akt, GSK3β, p-GSK3β, and β-catenin were detected by western blot. The bar graphs represent the band intensity compared to the α-MSH control. Data are represented as means ± SD.


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