Nutr Res Pract.  2014 Oct;8(5):509-515. 10.4162/nrp.2014.8.5.509.

Vitis amurensis Ruprecht root inhibited alpha-melanocyte stimulating hormone-induced melanogenesis in B16F10 cells

• Jin KS
• Oh YN
• Hyun SK
• Kwon HJ
• Kim BW

• KMID: 2313771
• DOI: http://doi.org/10.4162/nrp.2014.8.5.509

Abstract

BACKGROUND/OBJECTIVES
The root of Vitis amurensis Ruprecht, a sort of wild-growing grape, has been used in oriental medicine for treatment of skin ailments; however, its dermatological activity is not sufficiently understood. The aim of this study was to investigate tyrosinase inhibitory and anti-melanogenic activities of V. amurensis Ruprecht root methanol extract (VARM) in B16F10 mouse melanoma cells and to attempt to isolate and identify the active compound issued from VARM.
MATERIALS/METHODS
Anti-melanogenic activity of VARM was analyzed in alpha-melanocyte stimulating hormone (MSH)-stimulated B16F10 cells through evaluation of antioxidative activity as well as inhibited tyrosinase activity and melanin contents compared with those of kojic acid and arbutin. After anti-melanogenic analysis of VARM, serial fractionation, nuclear magnetic resonance (NMR), and thin layer chromatorgraphy (TLC) were applied for identification of active compounds contained in VARM.
RESULTS
VARM significantly inhibited oxidative stress and tyrosinase activity and attenuated alpha-MSH-induced melanin production in B16F10 cells. For isolation of active compounds, VARM was fractionated using a series of organic solvents, including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Among fractions showing anti-melanogenic activity, the CH2Cl2 fraction induced the most potent attenuation of melanogenesis without cytotoxicity and the major compound in the CH2Cl2 fraction was identified as betulinic acid. Betulinic acid isolated from the CH2Cl2 fraction of VARM significantly attenuated alpha-MSH-induced melanogenesis in a dose dependent manner, which was stronger than that of arbutin used as a positive control.
CONCLUSIONS
These results indicate that VARM inhibits oxidative stress, tyrosinase activity, and alpha-MSH-induced melanogenesis in B16F10 cells, due primarily to the active compound, betulinic acid, in the CH2Cl2 fraction.

Keyword

Vitis amurensis Ruprecht root; anti-oxidation; tyrosinase; melanogenesis; betulinic acid

MeSH Terms

1-Butanol
Animals
Arbutin
Magnetic Resonance Spectroscopy
Medicine, East Asian Traditional
Melanins
Melanoma
Methanol
Methylene Chloride
Mice
Monophenol Monooxygenase
Oxidative Stress
Skin
Solvents
Vitis*
1-Butanol
Arbutin
Melanins
Methanol
Methylene Chloride
Monophenol Monooxygenase
Solvents

Figure

• 

  Fig. 1 DPPH radical scavenging activity (A) and tyrosinase inhibition activity (B) of VARM. Ascorbic acid (C) and Kojic acid (D) are used as positive controls for DPPH radical scavenging activity and tyrosinase inhibition activity, respectively.

• 

  Fig. 2 Effect of VARM on B16F10 cell viability (A) and α-MSH induced melanogenesis (B). Arbutin is used as a positive control. Values are expressed as mean ± SD (n = 3). *, ** Significantly different from the vehicle treated control and α-MSH treated control, respectively (P < 0.05).

• 

  Fig. 3 Scheme of the preparation and isolation of VARM, its solvent fractions, and active compounds originated from the C fraction of VARM

• 

  Fig. 4 Effect of VARM and its solvent fractions on B16F10 cell viability (A) and α-MSH induced melanogenesis (B). M, VARM; C, CH2Cl2 fraction; E, EtOAc fraction; B, BuOH fraction; W, H2O fraction. Arbutin is used as a positive control. *, **Significantly different from the vehicle treated control and α-MSH treated control, respectively (P < 0.05).

• 

  Fig. 5 Effect of VARM and its solvent fractions on cellular tyrosinase activity (A) and tyrosinase protein expression (B). M, VARM; C, CH2Cl2 fraction; E, EtOAc fraction; B, BuOH fraction; A, arbutin used as a positive control. *, **Significantly different from the vehicle treated control and α-MSH treated control, respectively (P < 0.05).

• 

  Fig. 6 Anti-melanogenic activity of the C fraction (A) and betulinic acid originated from the C fraction (B) of VARM. Arbutin is used as a positive control. *, **Significantly different from the vehicle treated control and α-MSH treated control, respectively (P < 0.05).

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