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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 &mu, M 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-eIF2&alpha, 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.

A hallmark of Alzheimer's disease (AD) is the aggressive appearance of plaques of amyloid beta (Aβ) peptides, which result from the sequential cleavage of amyloid precursor protein (APP) by the β- and γ-secretases. Aβ 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 Aβ 1-42 peptide (Aβ42) and on the expressions of the related genes in SH-SY5Y human neuroblastoma cells. The treatment of cells with CA suppressed Aβ42 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 β-secretase BACE1 (β-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 Aβ42 secretion. These results suggest that CA reduces Aβ42 production by activating TACE without promoting BACE1 in human neuroblastoma cells. The use of CA may have a potential in the prevention of Aβ-mediated diseases, particularly AD.

Alzheimer’s disease (AD) is characterized by synaptic and neuronal loss, which occurs at least partially through oxidative stress induced by oligomeric amyloid-β (Aβ)-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 Aβ. 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 Aβ. 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, Aβ 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.

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.

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.

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 lightinduced 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, darkadapted 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 H2O2-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. (Invest Ophthalmol Vis Sci. 2012; 53:7847‐7854) DOI:10.1167/iovs.12-10793