title: Partially purified components of Nardostachys chinensis suppress melanin synthesis through ERK and Akt signaling pathway with cAMP down-regulation in B16F10 cells.
abstract: Ethnopharmacological relevance Nardostachys chinensis has been used in folk medicine to treat melasma and lentigines in Korea. We investigated the inhibitory activities of Nardostachys chinensis in melanogenesis and its related signaling pathway.
  
  
  METHODS
  Bioassay-guided fractionation of Nardostachys chinensis using solvent partitioning and purification with octadecylsilane open-column chromatography resulted in partial purification. The active 20% methanol chromatographic fraction from the ethyl acetate layer (PPNC) was used to investigate melanogenesis by melanin synthesis, tyrosinase activity assay, cAMP assay, Western blot and flow cytometric analyses in B16F10 mouse melanoma cells.
  
  
  RESULTS
  PPNC markedly inhibits melanin synthesis and tyrosinase activity in a concentration-dependent manner. We also found that PPNC decreases microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and dopachrome tautomerase (Dct) protein expressions and MITF and tyrosinase mRNA levels. Moreover, PPNC reduces intracellular cAMP levels and activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt expression in B16F10 cells. The specific MEK/ERK inhibitor PD98059 and PI3K/Akt inhibitor LY294002, block the PPNC-induced hypopigmentation effect, and abrogate the PPNC-suppressed expression of melanogenic proteins such as MITF, tyrosinase, TRP-1, and Dct. Using flow cytometry, we elucidated whether PPNC directly induces ERK phosphorylation at the level of an intact single cell. PPNC shows marked expression of phosphorylated ERK in live B16F10 cells and abrogates PPNC-induced phosphorylated ERK by PD98059 treatment.
  
  
  CONCLUSIONS
  PPNC stimulates MEK/ERK phosphorylation and PI3K/Akt signaling with suppressing cAMP levels and subsequently stimulating MITF and TRPs down-regulation, resulting in melanin synthesis suppression.
authors.affiliation: Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Gyeongnam, Republic of Korea.
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Jang
authors.forename: Ha Neui
authors.initials: HN
authors.lastname: Kim
authors.forename: Yu Ri
authors.initials: YR
authors.lastname: Kim
authors.forename: Woo Young
authors.initials: WY
authors.lastname: Choi
authors.forename: Yung Hyun
authors.initials: YH
authors.lastname: Choi
authors.forename: Hwa Kyoung
authors.initials: HK
authors.lastname: Shin
authors.forename: Byung Tae
authors.initials: BT
authors.lastname: Choi
mesh_terms: D000085:Acetates / Q000737:chemistry; D000818:Animals; D057886:Bleaching Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D015153:Blotting, Western; D005591:Chemical Fractionation / Q000379:methods; D002845:Chromatography; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008543:Melanins / Q000378:metabolism*; D008544:Melanocytes / Q000187:drug effects* / Q000201:enzymology; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000235:genetics; D000432:Methanol / Q000737:chemistry; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D032404:Nardostachys / Q000737:chemistry; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D012821:Silanes / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013997:Time Factors
chemical_list: D000085:Acetates; D057886:Bleaching Agents; D004365:Drugs, Chinese Herbal; D008543:Melanins; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D012821:Silanes; D012997:Solvents; C024779:octadecylsilane; C007650:ethyl acetate; D000242:Cyclic AMP; D014442:Monophenol Monooxygenase; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D000432:Methanol