Showing papers on "Carnosic acid published in 2017"

Carnosic acid and carnosol, two major antioxidants of rosemary, act through different mechanisms

Abstract: Carnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary (Rosmarinus officinalis). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis (Arabidopsis thaliana) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta.

Carnosic acid prevents dextran sulfate sodium-induced acute colitis associated with the regulation of the Keap1/Nrf2 pathway

Abstract: Crohn’s disease and ulcerative colitis are inflammatory bowel diseases (IBDs) with high prevalence in humans. Carnosic acid (CA) has been reported to possess antioxidative properties; however, its role in IBDs has not been determined. In the present study, we found that CA significantly prevented the loss of body weight and shortening of colon length in acute colitis induced by dextran sodium sulfate (DSS). Pronounced infiltration of immune cells and a loss of crypt architecture and goblet cells were ameliorated by CA. CA significantly decreased the activity of MPO and infiltration of F4/80+ macrophages in the colon. DSS-induced pro-inflammatory cytokine mRNA and protein levels in the colon were also attenuated by CA. CA decreased the activation of p65 and c-Jun signalling. CA inhibited DSS-induced NLRP3 inflammasome activation by reducing caspase 1 activity. In addition, CA increased the level of Nrf2 and prevented the degradation of Nrf2 via ubiquitination by blocking the interaction between Cullin3 and Keap1, which resulted in the decrease of Nrf2 target genes. Finally, GSH levels and SOD activity were increased after CA treatment, while MDA and iNOS levels were significantly reduced. Taken together, our data showed that CA may be useful as a potential therapeutic candidate for IBDs.

Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling

Abstract: Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor, which plays an important role in the cellular defense against oxidative stress by induction of anti-oxidant and cytoprotective enzymes. In the current study, we sought to investigate the osteoprotective effect of carnosic acid (CA), a phenolic (catecholic) diterpene. It is widely identified for its electrophilic nature under oxidative stress conditions and thus anticipated to counter osteoporosis by facilitation of Nrf2 signalling. Osteoclast differentiation was induced by incubation of RAW 264.7 (mouse macrophage) cells and mouse bone marrow macrophages (BMMs) in the presence of receptor activator of NF-κB ligand (RANKL) (100 ng/ml). After treatment, osteoclastogenesis was assessed using tartrate-resistant acid phosphatase (TRAP) assay. We observed that 6 h pretreatment with CA (1.25, 2.5, 5 μM) decreased RANKL-induced osteoclast formation and abolished RANKL-induced ROS generation by activating Nrf2 and its transcriptional targets. Further, CA also inhibited RANKL-induced activation of NF-κB and MAPK signalling. RANKL-induced mRNA expression of osteoclast related genes and transcription factors was also diminished by CA. In vivo osteolysis was developed in C57BL/6 male mice using lipopolysaccharide (LPS). Consistent with in vitro results, in vivo μ-CT analysis of femurs showed that bone mineral density (BMD), bone mineral content (BMC), and bone architecture parameters such as trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) were positively modulated by CA in LPS-injected mice. The results obtained in this study support that CA inhibits RANKL-induced osteoclastogenesis by maintaining redox homeostasis through modulation of Nrf2 and NF-κB pathways.

Anti-inflammatory and analgesic activity of carnosol and carnosic acid in vivo and in vitro and in silico analysis of their target interactions

Abstract: Background and Purpose The diterpenoids carnosol (CS) and carnosic acid (CA) from Salvia spp. exert prominent anti-inflammatory activities but their molecular mechanisms remained unclear. Here we investigated the effectiveness of CS and CA in inflammatory pain and the cellular interference with their putative molecular targets. Experimental Approach The effects of CS and CA in different models of inflammatory pain were investigated. The inhibition of key enzymes in eicosanoid biosynthesis, namely microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) was confirmed by CS and CA, and we determined the consequence on the eicosanoid network in activated human primary monocytes and neutrophils. Molecular interactions and binding modes of CS and CA to target enzymes were analyzed by docking studies. Key Results CS and CA displayed significant and dose-dependent anti-inflammatory and anti-nociceptive effects in carrageenan-induced mouse hyperalgesia 4 h post injection of the stimuli, and also inhibited the analgesic response in the late phase of the formalin test. Moreover, both compounds potently inhibited cell-free mPGES-1 and 5-LO activity and preferentially suppressed the formation of mPGES-1 and 5-LO-derived products in cellular studies. Our in silico analysis for mPGES-1 and 5-LO supports that CS and CA are dual 5-LO/mPGES-1 inhibitors. Conclusion and Implications In summary, we propose that the combined inhibition of mPGES-1 and 5-LO by CS and CA essentially contributes to the bioactivity of these diterpenoids. Our findings pave the way for a rational use of Salvia spp., traditionally used as anti-inflammatory remedy, in the continuous expanding context of nutraceuticals. Linked Articles This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc

Carnosic Acid Attenuates 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells by Inducing Autophagy Through an Enhanced Interaction of Parkin and Beclin1

Abstract: Enhanced removal of abnormal protein aggregates or injured organelles through autophagy is related to neuroprotection in Parkinson's disease. In this study, we explored whether the induction of autophagy is associated with the neuroprotection of rosemary carnosic acid (CA) against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. The results indicated that cells treated with CA had increased protein levels of parkin and autophagy-related markers, including phosphatidylinositol 3-kinase p100, Beclin1, autophagy-related gene 7, and microtubule-associated protein 1 light chain 3-II, as well as enhanced formation of autophagic vacuoles. Treatment of cells with 6-OHDA decreased the levels of parkin and the autophagy markers, but CA pretreatment reversed these effects. However, wortmannin (an autophagosome formation blocker) pretreatment attenuated the effect of CA. After CA pretreatment, the induction of cleaved caspase 3, cleaved poly-ADP ribose polymerase, and nuclear condensation by 6-OHDA were alleviated. Both wortmannin and bafilomycin A1 (an autophagosome-lysosome fusion blocker) inhibited the anti-apoptosis effects of CA. Additionally, we performed immunoprecipitation with anti-parkin antibody and found that the interaction of parkin and Beclin1 protein was reduced by 6-OHDA but that this effect was reversed in cells pretreated with CA. Moreover, transfection of parkin siRNA in cells inhibited the ability of CA to alleviate 6-OHDA-decreased autophagy-related markers and nuclear condensation. In conclusion, CA protects against 6-OHDA-induced apoptosis by inducing autophagy through the interaction of parkin and Beclin1. These results provide a future strategy for use of CA in the prevention of Parkinson's disease.

Carnosic Acid Protects Mitochondria of Human Neuroblastoma SH-SY5Y Cells Exposed to Paraquat Through Activation of the Nrf2/HO-1Axis.

Abstract: Carnosic acid (CA; C20H28O4), which is also called salvin, is a major phenolic diterpene found in Rosmarinus officinalis L. and exhibits antioxidant, anti-inflammatory, and antiproliferative properties. CA activates the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, leading to the upregulation of antioxidant and phase II detoxification enzymes, such as heme oxygenase-1 (HO-1), glutathione reductase (GR), γ-glutamate-cysteine ligase (γ-GCL), and glutathione S-transferase (GST), among others. We have previously demonstrated that CA upregulates the total and mitochondrial synthesis of glutathione (GSH), causing mitochondrial protection against paraquat (PQ) and methylglyoxal (MG). Nonetheless, the complete mechanism by which CA prevented mitochondrial dysfunction was not clear yet. Here, we examine whether HO-1 would be involved in the CA-induced mechanism of mitochondrial protection in SH-SY5Y-treated cells. SH-SY5Y cells were pretreated with CA (1 μM) for 12 h prior to a challenge with PQ at 100 μM for additional 24 h. Zinc protoporphyrin IX (ZnPP IX; a specific inhibitor of HO-1; 10 μM) was utilized prior to exposure to CA in order to investigate whether HO-1 was involved in the cytoprotective effects elicited by CA. We found that the CA-induced Nrf2-dependent HO-1 upregulation ameliorated, at least in part, the mitochondrial function in PQ-treated cells. Therefore, CA protected mitochondria of SH-SY5Y cells and exerted anti-apoptotic effects by activating the Nrf2/HO-1 axis.

Rosmarinic acid potentiates carnosic acid induced apoptosis in lung fibroblasts.

Abstract: Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA) and rosmarinic acid (RA) was reported to cure bleomycin-(BLM)-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF) viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively) treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy) partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vitamine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts.

Cytotoxic responses of carnosic acid and doxorubicin on breast cancer cells in butterfly-shaped microchips in comparison to 2D and 3D culture.

Abstract: Two dimensional (2D) cell culture systems lack the ability to mimic in vivo conditions resulting in limitations for preclinical cell-based drug and toxicity screening assays and modelling tumor biology. Alternatively, 3D cell culture systems mimic the specificity of native tissue with better physiological integrity. In this regard, microfluidic chips have gained wide applicability for in vitro 3D cancer cell studies. The aim of this research was to develop a 3D biomimetic model comprising culture of breast cancer cells in butterfly-shaped microchip to determine the cytotoxicity of carnosic acid and doxorubicin on both estrogen dependent (MCF-7) and independent (MDA-MB231) breast cancer cells along with healthy mammary epithelial cells (MCF-10A) in 2D, 3D Matrigel™ and butterfly-shaped microchip environment. According to the developed mimetic model, carnosic acid exhibited a higher cytotoxicity towards MDA-MB 231, while doxorubicin was more effective against MCF-7. Although the cell viabilities were higher in comparison to 2D and 3D cell culture systems, the responses of the investigated molecules were different in the microchips based on the molecular weight and structural complexity indicating the importance of biomimicry in a physiologically relevant matrix.

Phytochemicals, Antioxidant and Antiproliferative Properties of Rosmarinus officinalis L on U937 and CaCo-2 Cells.

Abstract: Rosmarinus officinalis L., a medicinal herb from the labiates family, has been reported to have potential benefit in the treatment and prevention of several diseases. In particular its phenolics have demonstrated protective effects on various types of cancer through several mechanisms. The present study aimed to determine the effects of rosemary phenolic extracts on human cell functions, with particular regard to their anti-proliferative properties in three cell types U937, CaCo-2 and the peripheral blood mononuclear cells (PBMCs). The radical scavenging and Ferric reducing abilities of the extracts have been assessed as well as their cyto-toxicity and effects on cell cycle distribution and apoptosis. About 13 compounds were identified with dominance of rosmarinic acid in the methanolic extract and phenolic diterpens in the ethyl acetate fraction (Carnosol, Carnosic acid and methyl Carnosate). The total polyphenolic content was important in the first extract with 2.589 ± 0.005 g/100 g in gallic acid equivalent compared to 0.763 ± 0.005 g/100 g. The methanolic fraction displayed higher antioxidant activity (DPPHIC50: 0.510 mg/mL and FRAP: 1.714 ± 0.068 mmol Fe2+/g) while ethyl acetate showed pronounced antiproliferative effects (IC50: 14.85 ± 0.20µg/mL and 14.95 ± 2.32 µg/mL respectively for U937 and CaCo-2 cells). The anti-proliferative effect was associated with a cell cycle arrest in S phase for U937 (62% of the population at 5 µg/mL) with a concomitant decrease in G1 and G2/M phases. Tested extracts displayed in addition early apoptotic effects in U937 and late apoptosis in CaCo-2 cells. The obtained data indicate that the identified phenolics are at least partially responsible for the observed cytotoxicity.

Carnosic acid and fisetin combination therapy enhances inhibition of lung cancer through apoptosis induction

Abstract: Carnosic acid is a phenolic diterpene with anti-inflammation, anticancer, anti-bacterial, anti-diabetic, as well as neuroprotective properties, which is generated by many species from Lamiaceae family. Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally flavonoid is abundantly produced in different vegetables and fruits. Fisetin has been reported to have various positive biological effects, including anti-proliferative, anticancer, anti-oxidative and neuroprotective effects. Lung cancer is reported as the most common neoplasm in human world-wide. In the present study, the possible benefits of carnosic acid combined with fisetin on lung cancer in vitro and in vivo was explored. Carnosic acid and fisetin combination led to apoptosis in lung cancer cells. Caspase-3 signaling pathway was promoted in carnosic acid and fisetin co-treatment, which was accompanied by anti-apoptotic proteins of Bcl-2 and Bcl-xl decreasing and pro-apoptotic signals of Bax and Bad increasing. The death receptor (DR) of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was enhanced in carnosic acid and fisetin combined treatment. Furthermore, the mouse xenograft model in vivo suggested that carnosic acid and fisetin combined treatment inhibited lung cancer growth in comparison to the carnosic acid or fisetin monotherapy. This study supplies a novel therapy to induce apoptosis to inhibit lung cancer through caspase-3 activation.

Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation.

Abstract: Compounds that increase the activity of the energy sensor AMP-activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [3H]-2-deoxy-D-glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post-hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0 μM CA a response (226±9.62 % of control, p=0.001), similar to maximum insulin (201±7.86 % of control, p=0.001) and metformin (213±10.74 % of control, p=0.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo. This article is protected by copyright. All rights reserved.

Protective effects of carnosic acid against mitochondria‑mediated injury in H9c2 cardiomyocytes induced by hypoxia/reoxygenation

Abstract: Myocardial ischemia and reperfusion occurs in myocardial infarction. Timely reperfusion will exacerbate the injury through the mitochondria-mediated apoptosis in cardiomyocytes due to the accumulation of excessive reactive oxygen species (ROS). In order to identify novel therapeutic approaches, the cardioprotective effects of carnosic acid and the underlying mechanisms were investigated in the present study in H9c2 cardiomyocytes injured by hypoxia/reoxygenation in vitro. The viability of H9c2 cardiomyocytes was detected by MTT assay and further confirmed by the detection of intracellular lactate dehydrogenase (LDH) release. The mitochondrial function in H9c2 cardiomyocytes was evaluated using fluorescence methods. The proteins related to apoptosis, including caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were analyzed by western blot analysis, and the activity of caspase-3 was determined using a colorimetric method. As a result, carnosic acid was demonstrated to improve the viability of H9c2 cardiomyocytes and suppress the leakage of cytosolic lactate dehydrogenase under hypoxia/reoxygenation. In addition, the overproduction of intracellular ROS and intracellular calcium overload were ameliorated in the presence of carnosic acid. The dysfunction of mitochondria in H9c2 cardiomyocytes was also attenuated by carnosic acid through blocking the collapse of mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening. Furthermore, the apoptosis of H9c2 cardiomyocytes resulted from hypoxia/reoxygenation was inhibited by carnosic acid through the upregulation of Bcl-2 and the downregulation of Bax and caspase-3 levels. These results provided evidence for further investigation that would assist in the development of novel therapeutic approaches for myocardial infarction.

Enhancing the efficacy of AREDS antioxidants in light-induced retinal degeneration

Abstract: Purpose Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage. Methods Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation. Results Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions. Conclusions Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.

Optimization of antioxidant efficacy of a deflavored and decolorized rosemary extract: effect of carnosol content on the oxidative stability of paprika colored beef patties.

Abstract: Considering the significance of natural antioxidants to preserve meat, the present study was undertaken to evaluate the efficacy of a deflavored and decolorised extract of rosemary (StabilRose™) for the production and preservation of naturally colored fresh meat. Oxidative rancidity of meat and color degradation of paprika oleoresin were exploited as model systems and compared with butylated hydroxyanisole (BHA). The results showed similar efficacy for 3% carnosic acid extract and BHA, with further enhancement in efficacy with respect to the carnosic acid content. A synergetic antioxidant effect of carnosol on carnosic acid content was also noticed to an extent of 1:1 (w/w) ratio, and further increase in carnosol content showed no improvement in the antioxidant efficacy. Finally, stabilized paprika and optimized rosemary extract containing carnosic acid and carnosol in 1:1 (w/w) ratio was successfully applied to produce naturally colored meat suitable for storage at 4 ± 1 °C.

Overcoming the plasticity of plant specialized metabolism for selective diterpene production in yeast.

Abstract: Plants synthesize numerous specialized metabolites (also termed natural products) to mediate dynamic interactions with their surroundings. The complexity of plant specialized metabolism is the result of an inherent biosynthetic plasticity rooted in the substrate and product promiscuity of the enzymes involved. The pathway of carnosic acid-related diterpenes in rosemary and sage involves promiscuous cytochrome P450s whose combined activity results in a multitude of structurally related compounds. Some of these minor products, such as pisiferic acid and salviol, have established bioactivity, but their limited availability prevents further evaluation. Reconstructing carnosic acid biosynthesis in yeast achieved significant titers of the main compound but could not specifically yield the minor products. Specific production of pisiferic acid and salviol was achieved by restricting the promiscuity of a key enzyme, CYP76AH24, through a single-residue substitution (F112L). Coupled with additional metabolic engineering interventions, overall improvements of 24 and 14-fold for pisiferic acid and salviol, respectively, were obtained. These results provide an example of how synthetic biology can help navigating the complex landscape of plant natural product biosynthesis to achieve heterologous production of useful minor metabolites. In the context of plant adaptation, these findings also suggest a molecular basis for the rapid evolution of terpene biosynthetic pathways.

Selenium supplementation into diets containing carnosic acid, fish and rapeseed oils affects the chemical profile of whole blood in lambs

Abstract: Whole blood consists of plasma (~54%) and various types of circulating cells (~46%) with a very low percentage of cells other than erythrocytes (Jones and Allison, 2007). The main ester-lipid components of blood are triacylglycerols, cholesterol esters and glycerophospholipids. The concentrations of essential elements (like Se, Zn, Cu, Co, Fe or Mg), fatty acids (FAs), vitamins or cholesterol in plasma and circulating blood cells as well as in other mammal tissues vary due to different physiological factors (i.e. dietary intake, intestinal absorption, metabolism and exchange among compartments) (Kincaid, 1999; Risé et al., 2007; Juniper et al., 2008; Niedźwiedzka et al., 2008; Czauderna et al., 2010). ABSTRACT. The concentration of macro and trace elements, fatty acids (FAs), vitamins, total cholesterol (TCh) in blood as well as in other tissues can be modulated by diet composition. Thus, the purpose of the present study was to evaluate the effects of fish oil (FO), carnosic acid (CA) and selenized-yeast (SeY) or selenate (SeVI) on concentration of FAs, TCh, α-tocopherol (αT) and selected elements in whole blood of lambs. Thirty male lambs were allocated into 5 groups of 6 animals each and fed for 35 days the following diets: control – basal diet (BD) with 3% rapeseed oil (RO), ROFO – BD with 2% RO and 1% FO, CA – BD with 2% RO, 1% FO and 0.1% CA, CASeY – BD with 2% RO, 1% FO, 0.1% CA and 0.35 mg Se as selenized-yeast (SeY) per kg of BD and CASeVI – BD with 2% RO, 1% FO, 0.1% CA and 0.35 mg Se as sodium selenate (SeVI) per kg of BD. In animals fed CASeVI diet the levels of saturated (SFAs), monoand polyunsaturated FAs, thrombogenic-SFAs and atherogenicSFAs decreased in comparison to the control group. On the other hand, in lambs fed CASeY diet the concentration of TCh in blood increased in comparison to lambs fed CA and CASeVI diets. Moreover, feeding CASeY diet also enhanced the concentration of αT in blood as compared to the animals fed ROFO and CASeVI diets. The lowest αT concentration in blood was noted in blood of lambs fed CASeVI diet. Feeding diets supplemented with SeY or SeVI increased the concentrations of Se and malondialdehyde in blood in comparison to other diets. So, the whole blood can be treated as the valuable non-invasive marker for evaluation of ruminant health status and nutritional quality of ruminant feeds. Received: 2 March 2017 Revised: 5 June 2017 Accepted: 8 September 2017

One‐step isolation of carnosic acid and carnosol from rosemary by centrifugal partition chromatography

Abstract: Carnosic acid and carnosol are the main bioactive components responsible for the significant antioxidant activity of Rosmarinus officinalis. Nevertheless, they are known for their instability in solutions. Separation of both compounds from crude rosemary extract was successfully achieved by one-step centrifugal partition chromatography without any degradation. A two-phase solvent system, hexane/ethyl acetate/methanol/water (3:2:3:2 v/v) was run on a preparative scale applying the elution-extrusion technique in descending mode. A 900 mg quantity of the crude extract containing 39.7% carnosic acid and 12.3% carnosol was loaded onto a 500 mL column, rotating at 1800 rpm. Carnosic acid and carnosol were obtained at purities of 96.1 ± 1% and 94.4 ± 0.9%, with recoveries of 94.3 ± 4.4% and 94.8 ± 2.3%, respectively. The compounds were identified by mass spectrometry, tandem mass spectrometry, and comparison with authentic standards.

Carnosic acid protects non-alcoholic fatty liver-induced dopaminergic neuron injury in rats

Abstract: Non-alcoholic fatty liver disease (NAFLD) has been reported to induce cognitive impairments of hippocampus and may influence central nervous system. In the present study, we investigated whether carnosic acid (CA) ameliorates dopaminergic neuron injury in a rat model of NAFLD. In order to induce NAFLD, rats were fed with high-fat diet (HFD) for 10 weeks. We found that continued CA administration reduced lipid accumulation marked by decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels, and an increase in high-density lipoprotein cholesterol (HDL-C) level in the serum. H&E staining revealed that feeding CA reduced lipid droplets accumulation, and alleviated oxidative stress by increasing in superoxide dismutase (SOD) level and decreasing in malondialdehyde (MDA) level in the liver. In addition, by measuring several parameters of gait analysis, we demonstrated that CA treatment ameliorated behavioral impairments, as evidenced by decreased duration and maximum variation, accompanied by increased average speed and cadence. Furthermore, CA treated-animals displayed an increase in the contents of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacelic acid (DOPAC) and elevated the expressions of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) as well as the TH protein in the striatum. Together, these findings suggest that CA may be an effective agent in protecting rats from NAFLD-induced dopaminergic neuron injury.

Carnosic acid, an inducer of NAD(P)H quinone oxidoreductase 1, enhances the cytotoxicity of β-lapachone in melanoma cell lines.

Abstract: NAD(P)H quinone oxidoreductase 1 (NQO1)-dependent antitumor drugs such as β-lapachone (β-lap) are attractive candidates for cancer chemotherapy because several tumors exhibit higher expression of NQO1 than adjacent tissues. Although the association between NQO1 and β-lap has been elucidated, the effects of a NQO1-inducer and β-lap used in combination remain to be clarified. It has previously been reported that melanoma cell lines have detectable levels of NQO1 expression and are sensitive to NQO1-dependent drugs such as 17-allylamino-17-demethoxygeldanamycin. The present study was conducted to investigate the involvement of NQO1 in β-lap-mediated toxicity and the utility of combination treatment with a NQO1-inducer and β-lap in malignant melanoma cell lines. Decreased expression or inhibition of NQO1 caused these cell lines to become less sensitive to β-lap, indicating a requirement of NQO1 activity for β-lap-mediated toxicity. Of note was that carnosic acid (CA), a compound extracted from rosemary, was able to induce further expression of NQO1 through NF-E2 related factor 2 (NRF2) stabilization, thus significantly enhancing the cytotoxicity of β-lap in all of the melanoma cell lines tested. Taken together, the data presented in the current study indicated that the NRF2-NQO1 axis may have potential value as a therapeutic target in malignant melanoma to improve the rate of clinical response to NQO1-dependent antitumor drugs.

Characterizing the effect of supplements on the phenotype of cultured macrophages from patients with age-related macular degeneration

Abstract: Purpose Oral vitamin and mineral supplements reduce the risk of visual loss in age-related macular degeneration (AMD). However, the pathways that mediate this beneficial effect are poorly understood. Macrophages may exert oxidative, inflammatory, and angiogenic effects in the context of AMD. We aim to assess if oral supplements can modulate the macrophage phenotype in this disease. Methods Monocytes were isolated from patients with neovascular AMD (nvAMD), cultured, matured to macrophages, and polarized to classical [M1 (stimulated by IFNγ and lipopolysaccharide (LPS))] and alternative [M2 (stimulated with IL-4 and IL-13)] phenotypes. Combinations of antioxidants including lutein+zeaxanthin (1 μM; 0.2 μM), zinc (10 µM), carnosic acid (2 µM), beta-carotene (2 µM), and standardized tomato extract containing lycopene and other tomato phytonutrients were added to the culture media. Levels of anti-inflammatory, antioxidant, and pro-angiogenic gene and protein expression were then evaluated. Results Combinations of lutein and carnosic acid with zinc and standardized tomato extract or with beta-carotene yielded an antioxidative, anti-inflammatory, and antiangiogenic effect in M1 and M2 macrophages. These effects manifested in the upregulation of antioxidative genes (HMOX1, SOD1) and the downregulation of pro-angiogenic genes and pro-inflammatory genes (SDF-1, TNF-alpha, IL-6, MCP-1). Lutein monotherapy or a combination of lutein and zinc had less effect on the expression of these genes. Conclusions Combinations of supplements can modify the expression of genes and proteins that may be relevant for the involvement of macrophages in the pathogenesis of AMD. Further studies are required to evaluate if the modulation of the macrophage phenotype partially accounts for the beneficial effect of oral supplements in AMD and if modification of the AREDS formula can improve its effect on macrophages.