Planta Med 2010; 76(7): 683-688
DOI: 10.1055/s-0029-1240622
Original Papers
© Georg Thieme Verlag KG Stuttgart · New York

Activated Glutathione Metabolism Participates in Protective Effects of Carnosic Acid against Oxidative Stress in Neuronal HT22 Cells

Yosei Tamaki1 , Takahito Tabuchi1 , Toshiyuki Takahashi1 , Kunio Kosaka2 , Takumi Satoh1
  • 1Department of Welfare Engineering, Faculty of Engineering, Iwate University, Morioka, Iwate, Japan
  • 2Research and Development Center, Nagase Co., Ltd., Kobe, Hyogo, Japan
Further Information

Publication History

received August 7, 2009 revised October 19, 2009

accepted October 26, 2009

Publication Date:
25 November 2009 (online)


In our previous studies, we have reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protects cortical neurons by inducing phase 2 enzymes, the induction of which was initiated by activation of the Keap1/Nrf2 pathway [1], [2], [3]. In the present study we address the nature of the effector of these neuroprotective effects downstream of the phase 2 enzyme induction. From our results we conclude that activated glutathione (GSH) metabolism may participate in these protective effects. First, we performed cDNA microarray analysis in order to identify the gene(s) responsible for the actions and found that various enzymes involved in the metabolism of GSH (glutathione S-transferase, alpha 4; glutathione S-transferase, alpha 2; and formylglutathione hydrolase) constituted 3 of the top 5 CA-induced genes. The other 2 genes encoded phase 2 enzymes [NAD(P)H-quinone oxidoreductase1and aldehyde dehydrogenase family 3, subfamily A1]. Next, we compared the physiologically-active compounds originating from rosemary (CA, CS, luteolin, genkwanin, rosmarinic acid, caffeic acid, and verbenone) by 3 criteria (enhancement of total glutathione levels, transcriptional activation, neuroprotective effects). By all of these criteria, CA and CS were the most active. In contrast, the other compounds were only weakly active or totally inactive. These results suggest that pro-electrophilic compounds such as CA and CS may protect cortical neurons by causing the following sequential events: S-alkylation → activation of the Keap1/Nrf2 pathway → transcriptional activation → induction of phase 2 enzymes → activation of GSH metabolism → neuroprotection.


  • 1 Satoh T, Kosaka K, Itoh K, Kobayashi A, Yamamoto M, Shimojo Y, Kitajima C, Cui J, Kamins J, Okamoto S, Shirasawa T, Lipton S A. Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of specific cysteines.  J Neurochem. 2008;  104 1116-1131
  • 2 Satoh T, Izumi M, Inukai Y, Tsutumi Y, Nakayama N, Kosaka K, Kitajima C, Itoh K, Yokoi T, Shirasawa T. Carnosic acid protects neuronal HT22 cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.  Neurosci Lett. 2008;  434 260-265
  • 3 Satoh T, Saitoh S, Hosaka H, Kosaka K. Simple ortho- and para-hydroquinones as neuroprotective compounds against oxidative stress associated with a specific transcriptional activation.  Biochem Biophys Res Commun. 2009;  379 537-541
  • 4 Kim S J, Kim J S, Cho H S, Lee H J, Kim S Y, Kim S, Lee S Y, Chun H S. Carnosol, a component of rosemary (Rosemarinus officinalis L.) protects nigral dopaminergic neuronal cells.  Neuroreport. 2006;  17 1729-1733
  • 5 Aruoma O I, Halliwell B, Aeschbach R, Lolingers J. Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid.  Xenobiotica. 1992;  22 257-268
  • 6 Martin D, Rojo A I, Salinas M, Diaz R, Gallardo G, Alam J, De Galarreta C M, Cuadrado A. Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol.  J Biol Chem. 2004;  279 8919-8929
  • 7 Rau O, Wurglics M, Paulke A, Zitzkowski J, Meindl N, Bock A, Dingermann T, Abdel-Tawab M, Schubert-Zsilavvecz M. Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs rosemary and sage, are activators of the human peroxisome proliferator-activated receptor gamma.  Planta Med. 2006;  72 881-887
  • 8 Visanji J M, Thompson D G, Padfield P J. Induction of G2/M phase cell cycle arrest by carnosol and carnosic acid is associated with alteration of cyclin A and cyclin B1 levels.  Cancer Lett. 2006;  237 130-136
  • 9 Subbaramaiah K, Cole P A, Dannenberg A J. Retinoid and carnosol suppress cyclooxygenase-2 transcription by CREB-binding protein/p 300-dependent and -independent mechanism.  Cancer Res. 2002;  62 2522-2530
  • 10 Huang S C, Ho C T, Lin-Shiau S Y, Lin J K. Carnosol inhibits the invasion of B16/F10 mouse melanoma cells by suppressing metalloproteinase-9 through down-regulating nuclear factor-kappa B and c-jun.  Biochem Pharmacol. 2005;  69 221-232
  • 11 Kosaka K, Yokoi T. Carnosic acid, a component of rosemary (Rosmarinus officinalis L.), promotes synthesis of nerve growth factor in T98 G human glioblastoma cells.  Biol Pharm Bull. 2003;  26 1620-1622
  • 12 Kraft A D, Johnson D A, Johnson J A. Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult.  J Neurosci. 2004;  24 1101-1112
  • 13 Sun X, Erb H, Murphy T H. Coordinate regulation of glutathione metabolism in astrocytes by Nrf2.  Biochem Biophys Res Commun. 2005;  326 371-377
  • 14 Satoh T, Lipton S A. Redox regulation of neuronal survival by electrophilic compounds.  Trends Neurosci. 2007;  30 37-45
  • 15 Satoh T, Okamoto S, Cui J, Watanabe Y, Furuta K, Suzuki M, Tohyama K, Lipton S A. Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers.  Proc Natl Acad Sci USA. 2006;  103 768-773
  • 16 Lipton S A. Pathologically-activated therapeutics for neuroprotection.  Nat Rev Neurosci. 2007;  8 803-808
  • 17 Talalay P. Chemoprotection against cancer by induction of phase 2 enzymes.  Biofactors. 2000;  12 5-11
  • 18 Itoh K, Tong K I, Yamamoto M. Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles.  Free Radic Biol Med. 2004;  36 1208-1213
  • 19 Wakabayashi N, Itoh K, Wakabayashi J, Motohashi H, Noda S, Takahashi S, Imakado T, Kotsuji T, Otsuka F, Roop D R, Harada T, Engel D J, Yamamoto M. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation.  Nat Genet. 2003;  35 238-245
  • 20 Satoh T, Harada N, Hosoya T, Ito K. Keap1/Nrf2 system regulates neuronal survival as revealed through study of keap1 gene knockout mice.  Biochem Biophys Res Commun. 2009;  380 298-302
  • 21 Takahashi T, Tabuchi T, Tamaki Y, Kosaka K, Satoh T. Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism.  Biochem Biophys Res Commun. 2009;  382 549-554
  • 22 Davis J B, Maher P. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line.  Brain Res. 1994;  652 169-173
  • 23 Coyle J T, Puttfarcken P. Oxidative stress, glutamate and neurodegenerative disorders.  Science. 1993;  262 689-695
  • 24 Satoh T, Furuta K, Tomokiyo K, Nakatsuka D, Miura M, Hatanaka H, Ikuta K, Suzuki M, Watanabe Y. Facilitatory roles of novel compounds designed from cyclopentenone prostaglandins on the neurite outgrowth-promoting activities of NGF.  J Neurochem. 2000;  75 1092-1102
  • 25 Satoh T, Furuta K, Tomokiyo K, Namura S, Nakatsuka D, Sugie Y, Ishikawa Y, Hatanaka H, Suzuki M, Watanabe Y. Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins.  J Neurochem. 2001;  77 50-62
  • 26 Satoh T, Baba M, Nakatsuka D, Ishikawa Y, Aburatani H, Furuta K, Ishikawa T, Hatanaka H, Suzuki M, Watanabe Y. Role of heme oxygenase-1 protein in the neuroprotective effects by cyclopentenone prostaglandin derivatives as a sustained phase of neuronal survival promoting mechanism under oxidative stress.  Eur J Neurosci. 2003;  17 2249-2255
  • 27 Uchida K. 4-Hydroxy 2-nonenal: a product and mediator of oxidative stress.  Prog Lipid Res. 2003;  42 318-343

Prof. Dr. Takumi Satoh

Department of Welfare Engineering
Faculty of Engineering
Iwate University

Ueda 4–3–5


020–8551 Iwate


Phone: + 81 1 96 21 60 39

Fax: + 81 1 96 21 63 14