Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes.

: Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 µM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert-butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.

Therapeutic advantage of pro-electrophilic drugs to activate the Nrf2/ARE pathway in Alzheimer's disease models.

Alzheimer's disease (AD) is characterized by synaptic and neuronal loss, which occurs at least partially through oxidative stress induced by oligomeric amyloid-ß (Aß)-peptide. Carnosic acid (CA), a chemical found in rosemary and sage, is a pro-electrophilic compound that is converted to its active form by oxidative stress. The active form stimulates the Keap1/Nrf2 transcriptional pathway and thus production of phase 2 antioxidant enzymes. We used both in vitro and in vivo models. For in vitro studies, we evaluated protective effects of CA on primary neurons exposed to oligomeric Aß. For in vivo studies, we used two transgenic mouse models of AD, human amyloid precursor protein (hAPP)-J20 mice and triple transgenic (3xTg AD) mice. We treated these mice trans-nasally with CA twice weekly for 3 months. Subsequently, we performed neurobehavioral tests and quantitative immunohistochemistry to assess effects on AD-related phenotypes, including learning and memory, and synaptic damage. In vitro, CA reduced dendritic spine loss in rat neurons exposed to oligomeric Aß. In vivo, CA treatment of hAPP-J20 mice improved learning and memory in the Morris water maze test. Histologically, CA increased dendritic and synaptic markers, and decreased astrogliosis, Aß plaque number, and phospho-tau staining in the hippocampus. We conclude that CA exhibits therapeutic benefits in rodent AD models and since the FDA has placed CA on the 'generally regarded as safe' (GRAS) list, thus obviating the need for safety studies, human clinical trials will be greatly expedited.

Carnosic acid attenuates apoptosis induced by amyloid-ß 1-42 or 1-43 in SH-SY5Y human neuroblastoma cells.

Amyloid-beta (Aß) peptides, Aß 1-42 (Aß42) and Aß43 in particular, cause neurotoxicity and cell death in the brain of Alzheimer's disease (AD) at higher concentrations. Carnosic acid (CA), a phenolic diterpene compound in the labiate herbs rosemary and sage, serves as an activator for neuroprotective and neurotrophic functions in brain cells. We investigated the effect of CA on apoptosis induced by Aß42 or Aß43 in cultured SH-SY5Y human neuroblastoma cells. Treatment of the cells with Aß42 or Aß43 (monomer, 10 µM each) induced apoptosis, which was confirmed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF). Concurrently, the Aß treatment induced the activation of caspase (Casp) cascades including an effector Casp (Casp3) and initiator Casps (Casp4, Casp8 and Casp9). Pretreatment of the cells with CA (10 µM) partially attenuated the apoptosis induced by Aß42 or Aß43. CA pretreatment also reduced the cellular oligomers of Aß42 and Aß43. These results suggest that CA suppressed the activation of Casp cascades by reducing the intracellular oligomerization of exogenous Aß42/43 monomer. The ingestion of an adequate amount of CA may have a potential in the prevention of Aß-mediated diseases, particularly AD.

Carnosic acid suppresses the production of amyloid-ß 1-42 and 1-43 by inducing an α-secretase TACE/ADAM17 in U373MG human astrocytoma cells.

Amyloid beta (Aß) peptides are key molecules in the pathogenesis of Alzheimer's disease (AD). The sequential cleavage of amyloid precursor protein (APP) by the ß- and γ-secretases generates Aß peptides; however, the alternate cleavage of APP by the α- and γ-secretases decreases Aß production. We previously reported that carnosic acid (CA), a phenolic diterpene compound found in the labiate herbs rosemary and sage, suppresses Aß (1-40 and 1-42) production by activating α-secretase in cultured SH-SY5Y human neuroblastoma cells (Neurosci. Res. 2013; 75: 94-102). Here, we investigated the effect of CA on the production of Aß peptides (1-40, 1-42 and 1-43) in U373MG human astrocytoma cells. The treatment of cells with CA suppressed Aß40/42/43 release (55-71% decrease at 50µM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17); however, the ß-secretase BACE1 (ß-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα release enhanced by CA and partially recovered the CA-suppressed Aß40/42/43 release. These results suggest that CA reduces Aß production, at least partially, by activating TACE in human astroglial cells. The use of CA may have a potential in the prevention of Aß-mediated diseases.

Carnosic acid suppresses the production of amyloid-ß 1-42 by inducing the metalloprotease gene TACE/ADAM17 in SH-SY5Y human neuroblastoma cells.

A hallmark of Alzheimer's disease (AD) is the aggressive appearance of plaques of amyloid beta (Aß) peptides, which result from the sequential cleavage of amyloid precursor protein (APP) by the ß- and γ-secretases. Aß production is evaded by alternate cleavage of APP by the α- and γ-secretases. Carnosic acid (CA) has been proven to activate the transcription factor Nrf2, a main regulator of the antioxidant response. We investigated the effects of CA on the production of Aß 1-42 peptide (Aß42) and on the expressions of the related genes in SH-SY5Y human neuroblastoma cells. The treatment of cells with CA suppressed Aß42 secretion (61% suppression at 30µM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17) significantly and another α-secretase ADAM10 marginally; however, the ß-secretase BACE1 (ß-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα secretion enhanced by CA and partially recovered the CA-suppressed Aß42 secretion. These results suggest that CA reduces Aß42 production by activating TACE without promoting BACE1 in human neuroblastoma cells. The use of CA may have a potential in the prevention of Aß-mediated diseases, particularly AD.

Protective effect of carnosic acid, a pro-electrophilic compound, in models of oxidative stress and light-induced retinal degeneration.

PURPOSE: The herb rosemary has been reported to have antioxidant and anti-inflammatory activity. We have previously shown that carnosic acid (CA), present in rosemary extract, crosses the blood-brain barrier to exert neuroprotective effects by upregulating endogenous antioxidant enzymes via the Nrf2 transcriptional pathway. Here we investigated the antioxidant and neuroprotective activity of CA in retinal cell lines exposed to oxidative stress and in a rat model of light-induced retinal degeneration (LIRD). METHODS: Retina-derived cell lines ARPE-19 and 661W treated with hydrogen peroxide were used as in vitro models for testing the protective activity of CA. For in vivo testing, dark-adapted rats were given intraperitoneal injections of CA prior to exposure to white light to assess protection of the photoreceptor cells. Retinal damage was assessed by measuring outer nuclear layer thickness and by electroretinogram (ERG). RESULTS: In vitro, CA significantly protected retina-derived cell lines (ARPE-19 and 661W) against H(2)O(2)-induced toxicity. CA induced antioxidant phase 2 enzymes and reduced formation of hyperoxidized peroxiredoxin (Prx)2. Similarly, we found that CA protected retinas in vivo from LIRD, producing significant improvement in outer nuclear layer thickness and ERG activity. CONCLUSIONS: These findings suggest that CA may potentially have clinical application to diseases affecting the outer retina, including age-related macular degeneration and retinitis pigmentosa, in which oxidative stress is thought to contribute to disease progression.

Carnosic acid (CA) prevents lipid accumulation in hepatocytes through the EGFR/MAPK pathway.

BACKGROUND: Carnosic acid (CA), found in rosemary, has been reported to have antioxidant and anti-adipogenic properties. We recently demonstrated that CA protects against steatosis in ob/ob mice. In the present report, we investigated the molecular mechanism by which CA inhibits lipids accumulation both in vivo and in vitro. METHODS: In the in vivo study, ob/ob mice were fed a standard chow diet with or without CA for 5 weeks, then their hepatocyte lipid accumulation was determined. The serum concentrations of cytokines, the levels of lipid regulatory mediators, and the hepatic metabolic and signaling molecules were also evaluated. In the in vitro study, HepG2 cells were used to further clarify the effects of CA on cellular lipid accumulation and to confirm the signaling pathways involved in these effects. RESULTS: CA significantly reduced hepatocyte lipid accumulation. This effect was associated with repressed levels of hepatic PPARγ, reduced expression of inflammatory cytokines such as IL-1ß, IL-12, IL-17, IFN-γ, MCP-1, and MIP-1ß, and increased ATP, acetyl CoA, NAD(P)(+), and NAD(P)H. Other signaling molecules, such as EGFR, MAPK, AMPK, and ACC, which regulate lipid metabolism, were activated in mice fed the CA diet. CA inhibited palmitate-induced cellular lipid accumulation and stimulated the phosphorylation of both EGFR and MAPK. Pretreatment with either the EGFR inhibitor AG1478 or the MEK-specific inhibitor U0126 abolished the effects of CA on cellular lipid accumulation and decreased both the protein expression and activity of PPARγ. CONCLUSIONS: EGFR/MAPK signaling plays an important role in the inhibitory effect of CA on hepatocyte lipid accumulation.

Nrf2 regulates NGF mRNA induction by carnosic acid in T98G glioblastoma cells and normal human astrocytes.

Nerve growth factor (NGF) is a neurotrophic factor that plays an important role in neuronal cell development and survival. Carnosic acid (CA), a hydrophobic constituent of the herb rosemary, induces NGF production in human T98G glioblastoma cells, but the mechanism through which it works remains unknown. In the present study, we found a redox-sensitive transcription factor, Nrf2, which coordinates the expression of cytoprotective phase 2 genes, also participates in CA-inducible NGF expression. In T98G cells, CA caused NGF gene induction in a dose- and time-dependent manner without altering NGF mRNA stability. Simultaneously, CA increased Nrf2 nuclear accumulation and activated expression of prototypical Nrf2 target genes such as haem oxygenase 1 (HO-1) and thioredoxin reductase 1 (TXNRD1). Knockdown of endogenous Nrf2 by Nrf2-specific siRNA significantly reduced constitutive and CA-inducible NGF gene expression. In addition, NGF gene expression was enhanced by knockdown of Keap1, an Nrf2 inhibitor, in the absence of CA. Furthermore, CA induced NGF expression in normal human astrocytes in an Nrf2-dependent manner. These results highlight a role of Nrf2 in NGF gene expression in astroglial cells.

Carnosic acid prevents obesity and hepatic steatosis in ob/ob mice.

AIM: Carnosic acid (CA) inhibits adipogenesis in vitro. The present study evaluated the therapeutic effects of CA in ob/ob mice. METHODS: The experimental animals were given a standard chow diet with or without CA for 5 weeks. Bodyweight gain and food intake were measured during this period. Magnetic resonance imaging analysis, histological examination, serum chemistry analysis and intraperitoneal glucose tolerance test (IPGTT) were all performed. RESULTS: The mice fed CA experienced significant weight loss and reduced visceral adiposity, in addition to significantly reduced serum triglyceride (TG) and cholesterol levels. Importantly, CA had a dramatic effect on the liver by reducing the hepatic TG content, thus decreasing serum alanine aminotransferase levels. In addition, IPGTT revealed that CA significantly improved glucose tolerance. CONCLUSION: These data suggest that CA is a novel therapeutic agent for obesity-related non-alcoholic fatty liver disease.

Edaravone and carnosic acid synergistically enhance the expression of nerve growth factor in human astrocytes under hypoxia/reoxygenation.

Edaravone is a brain-penetrant free radical scavenger that is known to ameliorate postischemic neuronal dysfunction. The transcription factor Nrf2 plays an important role in the coordinated expression of stress-inducible genes. Here we examined the effects of edaravone and carnosic acid (CA), an Nrf2-inducer, on the expression of nerve growth factor (NGF) in human astrocytes exposed to hypoxia/reoxygenation. Cultured astrocytes were exposed to hypoxia for up to 4.5 h and then treated with edaravone and/or CA under normoxia (reoxygenation) for up to 72 h. Edaravone (∼1 mM) and CA (∼50 µM) treatment synergistically enhanced NGF expression. Nrf2 knockdown by siRNA and the inhibition of JNK (c-Jun N-terminal kinase) by SP600125 decreased both CA-induced NGF expression and Nrf2 nuclear accumulation and suppressed their synergistic effect on NGF expression. In contrast, the MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor U0126 suppressed the synergism without inhibiting CA-induced NGF expression. These results suggest that the synergistic effects of CA and edaravone depend, at least partially, on JNK-dependent Nrf2 accumulation (induced by CA) and on MEK-dependent pathways (induced by edaravone). We conclude that the use of edaravone and CA in combination may have therapeutic potential in the treatment of brain damage, particularly ischemia/reperfusion injury.

Effect of Ganoderma lucidum extract on adipocyte differentiation and adiponectin gene expression in the murine pre-adipocyte cell line, 3T3-L1.

Ganoderma lucidum (G. lucidum), a traditional Chinese medicine, has been used for the treatment of various diseases including cancer and atherosclerosis. In this study, the positive effect of G. lucidum on metabolic syndrome was investigated in more detail by the use of 3T3-L1 pre-adipocyte cells. Treatment of 3T3-L1 cells with G. lucidum extract (GE) significantly promoted adipocyte differentiation and adiponectin production in a dose-dependent manner, as assessed by Oil-Red O staining, quantitative RT-PCR and ELISA. Treatment with GW9662, an inhibitor for peroxisome proliferator-activated receptor-gamma (PPARgamma), significantly attenuated GE-dependent adipocyte differentiation and adiponectin gene expression, suggesting the involvement of PPARgamma. Moreover, a reporter gene assay using GAL4-PPAR fusion proteins revealed that GE enhances GAL4-PPARgamma and GAL4-PPARalpha activities. These results indicate the presence of natural compounds possessing PPARgamma and PPARalpha activating properties in G. lucidum.

Activated glutathione metabolism participates in protective effects of carnosic acid against oxidative stress in neuronal HT22 cells.

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 , , . 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.

Effect of rosmarinic acid in motor dysfunction and life span in a mouse model of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease affecting motor neurons. About 2% of patients with the disease are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). The purpose of this study is to assess the effect of rosemary extract and its major constituents, rosmarinic acid (RA) and carnosic acid (CA), in human SOD1 G93A transgenic mice, which are well-established mouse models for ALS. The present study demonstrates that intraperitoneal administration of rosemary extract or RA from the presymptomatic stage significantly delayed motor dysfunction in paw grip endurance tests, attenuated the degeneration of motor neurons, and extended the life span of ALS model mice. In addition, RA administration significantly improved the clinical score and suppressed body weight loss compared with a vehicle-treated group. In conclusion, this study provides the first report that rosemary extract and, especially, RA have preventive effects in the mouse model of ALS.

Role of Nrf2 and p62/ZIP in the neurite outgrowth by carnosic acid in PC12h cells.

Neurotrophins such as NGF promote neuronal survival and differentiation via the cell surface TrkA neurotrophin receptor. Compounds with neurotrophic actions that are low in molecular weight and can permeate the blood-brain barrier are promising therapeutic agents against neurodegenerative diseases such as Alzheimer's disease. Carnosic acid (CA), an electrophilic compound in rosemary, activates antioxidant responsive element (ARE)-mediated transcription via activation of Nrf2. In the present study, we discovered that CA strongly promotes neurite outgrowth of PC12h cells. NGF as well as CA activated Nrf2, whereas CA and NGF-mediated neuronal differentiation was suppressed by Nrf2 knockdown. On the other hand, CA activated TrkA-downstream kinase Erk1/2 independently of Nrf2. CA-induced p62/ZIP expression in an Nrf2-dependent manner, while the CA-induced neural differentiation was suppressed by p62/ZIP knockdown. Furthermore, CA-induced ARE activation was attenuated both by p62/ZIP knockdown and a Trk signal inhibitor. These results suggest that the CA induction of p62/ZIP by Nrf2 enhances TrkA signaling which subsequently potentiates Nrf2 pathway. This is the first demonstration that activation of the Nrf2-p62/ZIP pathway by a low-molecular natural electrophilic compound plays important roles in TrkA-mediated neural differentiation and may represent the common molecular mechanism for neurotrophic activities of electrophilic compounds.

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism.

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.

Simple ortho- and para-hydroquinones as compounds neuroprotective against oxidative stress in a manner associated with specific transcriptional activation.

Electrophilic compounds protect neurons through the activation of the Keap1/Nrf2 pathway and the induction of phase-2 enzymes [T. Satoh, S.A. Lipton, Redox regulation of neuronal survival by electrophilic compounds, Trends Neurosci. 30 (2007) 38-45; T. Satoh, S. Okamoto, J. Cui, Y. Watanabe, K. Furuta, M. Suzuki, K. Tohyama, S.A. Lipton, Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers. Proc. Natl. Acad. Sci. USA 103 (2006) 768-773]. Hydroquinone-type electrophilic compounds such as tert-butyl hydroquinone (TBHQ) and carnosic acid (CA) have attracted special attention, because the oxidative conversion of "hydroquinone" to "quinone" is essential for the transcriptional activation of the above-mentioned enzymes [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protect neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of specific cysteine, J. Neurochem. 104 (2008) 1161-1131; A.D. Kraft, D.A. Johnson, J.A. Johnson, 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. 24 (2004) 1101-1112]. In the present study, we examined the relationship between electrophilicity and the protective effects afforded by electrophilic compounds. Electrophilicity was assessed in terms of the ability of a compound to bind to a cysteine on bovine serum albumin, by which we found that neuroprotective hydroquinones [TBHQ (para-) and CA (ortho-)] had distinctive patterns of cysteine binding compared with other electrophilic compounds. Further, we found that isomers of simple ortho- and para-hydroquinones such as 2-methylhydroquinone (para-) and 4-methyl-catechol (ortho-) [not in abstract] had similar properties of cysteine binding as TBHQ and CA, which compounds were associated with the transcriptional activation and an increase in the level of reduced glutathione. These results suggest that para- and ortho-dihydroquinones may be neuroprotective compounds active against oxidative stress.

Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, 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 targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

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 targeted cysteines on Keap1.

Electrophilic compounds are a newly recognized class of redox-active neuroprotective compounds with electron deficient, electrophilic carbon centers that react with specific cysteine residues on targeted proteins via thiol (S-)alkylation. Although plants produce a variety of physiologically active electrophilic compounds, the detailed mechanism of action of these compounds remains unknown. Catechol ring-containing compounds have attracted attention because they become electrophilic quinones upon oxidation, although they are not themselves electrophilic. In this study, we focused on the neuroprotective effects of one such compound, carnosic acid (CA), found in the herb rosemary obtained from Rosmarinus officinalis. We found that CA activates the Keap1/Nrf2 transcriptional pathway by binding to specific Keap1 cysteine residues, thus protecting neurons from oxidative stress and excitotoxicity. In cerebrocortical cultures, CA-biotin accumulates in non-neuronal cells at low concentrations and in neurons at higher concentrations. We present evidence that both the neuronal and non-neuronal distribution of CA may contribute to its neuroprotective effect. Furthermore, CA translocates into the brain, increases the level of reduced glutathione in vivo, and protects the brain against middle cerebral artery ischemia/reperfusion, suggesting that CA may represent a new type of neuroprotective electrophilic compound.

Byelyankacin: a novel melanogenesis inhibitor produced by Enterobacter sp. B20.

A novel melanogenesis inhibitor, byelyankacin (1), was isolated from the fermentation broth of a bacterial strain. The producing organism, designated B20, was identified as a member of the genus Enterobacter based on taxonomic characteristics. 1 was obtained as a white powder from the culture medium by solvent extraction and serial chromatographic purification. The structure of 1 was determined as (E)-4-(2-isocyanovinyl)phenyl alpha-L-rhamnopyranoside on the basis of spectroscopic data. 1 potently inhibited mushroom tyrosinase and melanogenesis of B16-2D2 melanoma cells with IC50 value of 2.1 nM and 30 nM, respectively.

Carnosic acid, a component of rosemary (Rosmarinus officinalis L.), promotes synthesis of nerve growth factor in T98G human glioblastoma cells.

Nerve growth factor (NGF) is a factor vital for the growth and functional maintenance of nerve tissue. The authors found that a rosemary (Rosmarinus officinalis L.) extract enhanced the production of NGF in T98G human glioblastoma cells. Furthermore, the results indicated that carnosic acid and carnosol, which are major components of the rosemary extract, were able to promote markedly enhanced synthesis of NGF.