carnosol and salvin

Diabetes mellitus is a chronic endocrine disease result from absolute or relative insulin secretion deficiency, insulin resistance, or both, and has become a major and growing public healthy menace worldwide. Currently, clinical antidiabetic drugs still have some limitations in efficacy and safety such as gastrointestinal side effects, hypoglycemia, or weight gain.

Topics: Abietanes; Animals; Anti-Inflammatory Agents; Antioxidants; Cinnamates; Depsides; Diabetes Mellitus; Glucose; Humans; Lipid Metabolism; Phenols; Phytotherapy; Plant Extracts; Rosmarinic Acid; Rosmarinus

Carnosic acid (CA) and carnosol are the major diterpenes found in Rosmarinus officinalis (rosemary), a culinary spice. CA and carnosol account for over 90 % of its anti-oxidant activity in rosemary leaves. The diterpenes exert anti-oxidant, anti-inflammatory, and anti-carcinogenic activities, and present neuroprotective effects in both in vitro and in vivo experimental models. In some cases, CA exerted protective effects upon neuronal cells more intensely than resveratrol or sulforaphane. Therefore, CA and carnosol demonstrate a potential pharmacological role for rosemary diterpenes in ameliorating mammalian brain redox status, among other parameters, as for instance the modulation of neuroinflammation. The aim of this review is to discuss the biological effects of CA and carnosol on neuronal and glial cells with focus on the mechanism of action of such diterpenes.

Topics: Abietanes; Animals; Diet; Humans; Neuroglia; Neurons; Neuroprotective Agents

Recently, rosemary extracts standardized to diterpenes (e.g. carnosic acid and carnosol) have been approved by the European Union (EU) and given a GRAS (Generally Recognized as Safe) status in the United States by the Food and Drug Administration (FDA). Incorporation of rosemary into our food system and through dietary selection (e.g. Mediterranean Diet) has increased the likelihood of exposure to diterpenes in rosemary. In consideration of this, a more thorough understanding of rosemary diterpenes is needed to understand its potential for a positive impact on human health. Three agents in particular have received the most attention that includes carnosic acid, carnosol, and rosmanol with promising results of anti-cancer activity. These studies have provided evidence of diterpenes to modulate deregulated signaling pathways in different solid and blood cancers. Rosemary extracts and the phytochemicals therein appear to be well tolerated in different animal models as evidenced by the extensive studies performed for approval by the EU and the FDA as an antioxidant food preservative. This mini-review reports on the pre-clinical studies performed with carnosic acid, carnosol, and rosmanol describing their mechanism of action in different cancers.

Topics: Abietanes; Animals; Antineoplastic Agents, Phytogenic; Diterpenes; Humans; Neoplasms; Phytotherapy; Plant Extracts; Plants, Medicinal; Rosmarinus; Signal Transduction

Cancer remains an important cause of mortality nowadays and, therefore, new therapeutic approaches are still needed. Rosemary (Rosmarinus officinalis L.) has been reported to possess antitumor activities both in vitro and in animal studies. Some of these activities were attributed to its major components, such as carnosic acid, carnosol, ursolic acid, and rosmarinic acid. Initially, the antitumor effects of rosemary were attributed to its antioxidant activity. However, in recent years, a lack of correlation between antioxidant and antitumor effects exerted by rosemary was reported, and different molecular mechanisms were related to its tumor inhibitory properties. Moreover, supported by the U.S. Food and Drug Administration and the European Food and Safety Authority, specific compositions of rosemary extract were demonstrated to be safe for human health and used as antioxidant additive in foods, suggesting the potential easy application of this agent as a complementary approach in cancer therapy. In this review, we aim to summarize the reported anticancer effects of rosemary, the demonstrated molecular mechanisms related to these effects and the interactions between rosemary and currently used anticancer agents. The possibility of using rosemary extract as a complementary agent in cancer therapy in comparison with its isolated components is discussed.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Breast Neoplasms; Cinnamates; Depsides; Drug Interactions; Europe; Humans; Neoplasms; Phytotherapy; Plant Extracts; Rosmarinic Acid; Rosmarinus; Triterpenes; United States; United States Food and Drug Administration; Ursolic Acid

An overview of recent advances in nuclear magnetic resonance (NMR) coupled with separation technologies and their application in natural product analysis is given and discussed. The different modes of LC-NMR operation are described, as well as how technical improvements assist in establishing LC-NMR as an important tool in the analysis of plant-derived compounds. On-flow, stopped-flow and loop-storage procedures are mentioned, together with the new LC-SPE-NMR configuration. The implementation of mass spectrometry in LC-NMR is also useful on account of the molecular weight and fragmentation information that it provides, especially when new plant species are studied. Cryogenic technology and capillary LC-NMR are the other important recent developments. Since the plant kingdom is endless in producing potential drug candidates, development and optimization of LC-NMR techniques convert the study of natural products to a less-time-consuming task, speeding up identification.

Topics: Abietanes; Biological Products; Chromatography, Liquid; Cinnamates; Depsides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Microchemistry; Phenanthrenes; Plant Extracts; Reproducibility of Results; Rosmarinic Acid; Sensitivity and Specificity

Ninety-five rosemary (Salvia rosmarinus Schleid.) genotypes, representing 24 wild populations, collected in different geographical areas in Italy and then cultivated under homogeneous environmental conditions, were characterised for volatile organic compounds (VOCs) and three main non-volatile phenolic diterpenes (carnosic acid, carnosol) and acids (rosmarinic acid). Cluster analysis of chemical data highlighted the occurrence of three main groups of populations: one with high levels of verbenone, α-pinene, bornyl acetate, carnosic acid and carnosol, a second one with relatively high levels of camphor and a third one with high levels of 1,8-cineole, β-pinene and sesquiterpenes. This clustering was consistent with geographic origin and previous genetic characterisation of the same populations. Correlation analysis suggested that levels of the key antioxidant carnosic acid were associated to levels of the key aroma compound verbenone. The observed diversity may be exploited in breeding programs to develop lines and designing products with improved flavour quality and functional properties.

Topics: Odorants; Plant Breeding; Rosmarinus; Salvia; Volatile Organic Compounds

The aim of the study was to examine the effect of ultrasonic maceration (U) on the extraction of carnosic acid (CA) and its derivative-carnosol (C)-directly from sage into fish oil, compared to homogenization-assisted maceration (H). It was shown that the ultrasonic maceration process (U) allowed for obtaining a macerate enriched in carnosic acid (CA) and carnosol (C), also containing rosmarinic acid (RA), total polyphenols, and plant pigments, and showing antioxidant properties (DPPH test). There was no unequivocal difference in the efficiency of extracting ingredients from sage into the oil macerate between U and H, with the use of ultrasound in most cases resulting in a greater extraction of C and less extraction of pigments from sage into the macerate than in H. The highest simultaneous contents of CA (147.5 mg/100 g) and C (42.7 mg/100 g) in the macerate were obtained after 60 min of maceration U when using a higher power (320 W). The amount of determined compounds also depended on the concentration of methanol (methanol; 70% methanol) used for the analysis. The maceration U is a simple, safe, "green method" of obtaining active substances, with a reduced number of steps, enabling an interesting application form of CA and C, e.g., for food or cosmetics.

Topics: Fish Oils; Methanol; Postmortem Changes; Salvia officinalis

A high degree of selectivity is required during the plant extraction process in order to obtain extracts enriched in specific compounds or to avoid the extraction of unwanted ones. Rosemary is well known for its antioxidant compounds (carnosic acid, carnosol and rosmarinic acid). The plant also contains pigments (i.e. carotenoids, chlorophylls) which may cause a colour problem during the use of the extract in cosmetic formulations, for example. Supercritical fluid extraction is considered as a selective technique for plant extraction. Due to the physico-chemical properties of supercritical fluids, related to pressure, temperature and modifier addition, it is possible to carry out sequential extraction with successive conditions to collect different fractions that are rich either in pigments or in bioactive compounds. The aim of this study was to selectively extract bioactive compounds (i.e. carnosic acid and rosmarinic acid) and pigments (carotenoids and chlorophylls) from rosemary using supercritical fluid extraction. The optimisation of the extraction method was carried out using supercritical fluid extraction online coupled with a supercritical fluid chromatography (SFE-SFC) system. Two columns of different polarities were coupled to achieve the separation of the targeted compounds every five minutes during the extraction. Four fractions were obtained: a first one rich in carotenoids obtained with pure CO2 (25°C and 20 MPa), a second rich in carnosic acid obtained with 3% polar modifier (EtOH:water 50/50 v/v), a third fraction rich in rosmarinic acid using 10% of the same modifier and a fourth fraction rich in chlorophylls with 30% of ethanol as modifier. These four samples were then analysed by UHPLC-DAD-ESI-QTOF-HRMS in order to identify other extracted compounds and to study how the selected conditions impacted their extraction.

Topics: Abietanes; Antioxidants; Carotenoids; Chlorophyll; Chromatography, High Pressure Liquid; Chromatography, Supercritical Fluid; Cinnamates; Depsides; Kinetics; Plant Extracts; Reference Standards; Rosmarinic Acid; Rosmarinus

Rosemary extract (RE) is an approved food preservative in the European Union and contains dietary phytochemicals that are beneficial for gastrointestinal health. This study investigated the effects of RE on dextran sodium sulfate (DSS)-induced colitis and also determined the pharmacokinetics of dietary phytochemicals administered to mice via oral gavage. Individual components of rosemary extract were separated and identified by LC-MS/MS. The pharmacokinetics of two major diterpenes from RE, carnosic acid (CA) and carnosol (CL), administered to mice via oral gavage were determined. Then, the effect of RE pre-treatment on the disease activity index (DAI) of DSS-induced colitis in mice was investigated. The study determined that 100 mg/kg RE significantly improved DAI in DSS-induced colitis compared to negative control. Sestrin 2 protein expression, which increased with DSS exposure, was reduced with RE treatment. Intestinal barrier integrity was also shown to improve via fluorescein isothiocyanate (FITC)-dextran administration and Western blot of zonula occludens-1 (ZO-1), a tight junction protein. Rosemary extract was able to improve the DAI of DSS-induced colitis in mice at a daily dose of 100 mg/kg and showed improvement in the intestinal barrier integrity. This study suggests that RE can be an effective preventative agent against IBD.

Topics: Abietanes; Administration, Oral; Animals; Colitis; Dextran Sulfate; Gene Expression Regulation; HT29 Cells; Humans; Male; Mice; Plant Extracts; Rosmarinus; Zonula Occludens-1 Protein

Carnosic acid (CA), carnosol (CL) and rosmarinic acid (RA), components of the herb rosemary, reportedly exert favorable metabolic actions. This study showed that both CA and CL, but not RA, induce significant phosphorylation of AMP-dependent kinase (AMPK) and its downstream acetyl-CoA carboxylase 1 (ACC1) in HepG2 hepatoma cells. Glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1), rate-limiting enzymes of hepatic gluconeogenesis, are upregulated by forskolin stimulation, and this upregulation was suppressed when incubated with CA or CL. Similarly, a forskolin-induced increase in CRE transcriptional activity involved in G6PC and PCK1 regulations was also stymied when incubated with CA or CL. In addition, mRNA levels of ACC1, fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) were significantly reduced when incubated with CA or CL. Finally, it was shown that CA and CL suppressed cell proliferation and reduced cell viability, possibly as a result of AMPK activation. These findings raise the possibility that CA and CL exert a protective effect against diabetes and fatty liver disease, as well as subsequent cases of hepatoma.

Topics: Abietanes; AMP-Activated Protein Kinases; Animals; Apoptosis; Cell Proliferation; Fatty Acids; Gene Expression Regulation; Gluconeogenesis; HEK293 Cells; Hep G2 Cells; Humans; Lipogenesis; Mice; Oxidation-Reduction; Phosphorylation; Plant Extracts; Rosmarinus; Tumor Suppressor Protein p53; Up-Regulation

The antiradical power, at equal concentrations of active principles, of the following antioxidants were studied using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay: butylated-hydroxyanisole, butylated-hydroxytoluene, tert-butylhydroquinone, ascorbyl palmitate, tocopherol, grape seed extract, olive extract and five rosemary extracts with different concentrations of carnosic acid (CA) and carnosol (COL). The reaction kinetics of DPPH scavenging activity in each studied substance identified significant variations in the time needed to reach the steady state. Rosemary extracts were seen to be more effective than the other compounds. CA had higher antioxidant activity than COL, although COL seemed to react faster with DPPH. The relevance of the CA/COL ratio for the antioxidant activity of rosemary extracts was also analysed. The presence of COL in rosemary extracts increased the antioxidant activity with an optimal CA/COL ratio of 2.5-3.0. Olive extract and grape seed extract seem to be very promising additives for use as technological antioxidants.

Topics: Abietanes; Antioxidants; Ascorbic Acid; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Food Additives; Plant Extracts; Rosmarinus; Tocopherols

Carnosol and carnosic acid are polyphenolic compounds found in rosemary and sage with known anti-oxidant, anti-inflammatory, and anti-microbial properties. Here, we addressed the potential use of carnosol and carnosic acid for in vitro bone tissue engineering applications, specifically depending on their cytotoxic effects on bone marrow stromal and stem cells, and osteosarcoma cells in monolayer and 3D cultures. Carnosol and carnosic acid displayed a bacteriostatic effect on Gram-positive bacteria, especially on S. aureus. The viability results indicated that bone marrow stromal cells and bone marrow stem cells were more tolerant to the presence of carnosol compared to osteosarcoma cells. 3D culture conditions increased this tolerance further for healthy cells, while not affecting the cytotoxic potential of carnosol for osteosarcoma cells. Carnosic acid was found to be more cytotoxic for all cell types used in the study. Results suggest that phenolic compounds might have potential use as anti-microbial and anti-carcinogenic agents for bone tissue engineering with further optimization for controlled release.

Topics: Abietanes; Animals; Anti-Infective Agents; Anticarcinogenic Agents; Bone Neoplasms; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Humans; Limb Salvage; Mesenchymal Stem Cells; Mice; Osteosarcoma; Tissue Engineering

The safety, efficacy and stability of natural antioxidants have been the focus of research in the food industry, with the aim of rapidly analyzing and controlling the quality of rosemary and its extracts, a novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous determination of rosmarinic acid, carnosol, carnosic acid, oleanolic acid and ursolic acid in rosemary. Chromatographic separation was conducted with gradient elution mode by using a Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm) with mobile phases of methanol and 0.6% acetic acid. The drift tube temperature of ELSD was 70 °C, and the pressure of nebulizer nitrogen gas was 40 Psi. The method developed has high sensitivity (with limits of detection from 1.3 to 8.6 μg/mL), acceptable linearity over the tested concentrations (with correlation coefficients from 0.991 to 0.999), good repeatability (with intra- and inter-day CV less than 3.1% for all analytes) and satisfactory accuracy (with recovery between 95.5% and 100.8%). The method has been demonstrated as a powerful tool for the functional ingredients analysis and quality control of rosemary and its extracts in a cost- and time-effective manner.

Topics: Abietanes; Calibration; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Limit of Detection; Oleanolic Acid; Reproducibility of Results; Rosmarinic Acid; Scattering, Radiation; Triterpenes; Ursolic Acid

Lipid oxidation of fish oil enriched cow milk and soy milk supplemented with rosemary extract stored at 2 °C was studied. Both peroxide value and volatile secondary lipid oxidation products were determined to monitor the progress of lipid oxidation. Rosemary extract inhibited lipid oxidation in fish oil enriched cow milk. In contrast, soy milk samples having much higher unsaturated fatty acid content showed higher lipid oxidation stability compared to cow milk. Reduction in the content of chlorogenic acid during storage suggested that this compound may contribute to the lipid oxidation stability of fish oil enriched soy milk product. Total carnosic acid and carnosol concentration declined much faster in soy milk than in cow milk. It is suggested from the results that food components could have significant impact on the fate of bioactive antioxidant compounds in a specific food product during storage.

Topics: Abietanes; Animals; Antioxidants; Fish Oils; Food Storage; Food, Fortified; Lipids; Milk; Oxidation-Reduction; Plant Extracts; Rosmarinus; Soy Milk

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.. 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 E. 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.. 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.. 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.

Topics: Abietanes; Analgesics; Animals; Anti-Inflammatory Agents; Computer Simulation; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Inflammation; Lipoxygenase Inhibitors; Male; Mice; Molecular Docking Simulation; Monocytes; Neutrophils; Pain; Prostaglandin-E Synthases; Salvia

Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.

Topics: Abietanes; Chromatography, Liquid; Colonic Neoplasms; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Homeostasis; HT29 Cells; Humans; Isotope Labeling; Proteasome Endopeptidase Complex; Proteomics; Signal Transduction; Tandem Mass Spectrometry; Unfolded Protein Response

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.

Topics: Abietanes; Chromatography; Plant Extracts; Rosmarinus

Pregnane X receptor (PXR) regulates the expression of many genes, including those involved in drug metabolism and transport, and has been linked to various diseases, including inflammatory bowel disease. In the present study, we determined whether carnosic acid and other chemicals in rosemary extract (carnosol, ursolic acid, and rosmarinic acid) are PXR activators. As assessed in dual-luciferase reporter gene assays, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, activated human PXR (hPXR) and mouse PXR (mPXR), whereas carnosol and ursolic acid, but not carnosic acid or rosmarinic acid, activated rat PXR (rPXR). Dose-response experiments indicated that carnosic acid, carnosol, and ursolic acid activated hPXR with EC

Topics: Abietanes; Animals; Cell Line; Cinnamates; Depsides; Gene Expression Regulation; Hep G2 Cells; Humans; Mice; Plant Extracts; Pregnane X Receptor; Rats; Receptors, Steroid; Rosmarinic Acid; Rosmarinus; Triterpenes; Ursolic Acid

Cosmetic industries focus on developing materials and resources that regulate skin pigmentation. Melanin, the major pigment in human skin, protects the skin against damage from ultraviolet light. An ethanolic extract of the leaves of Callicarpa longissima inhibits melanin production in B16F10 mouse melanoma cells by suppressing microphthalmia-associated transcription factor (MITF) gene expression. Following purification and analysis using liquid chromatography-mass spectrometry (LC-MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of the C. longissima extract on melanin production. Carnosol is an oxidative product of carnosic acid, whose presence in the extract was also confirmed by an authentic reference. The carnosol and carnosic acid content in the extract was approximately 16% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents.

Topics: Abietanes; Animals; Callicarpa; Cell Differentiation; Cell Line, Tumor; Chromatography, Liquid; Gene Expression; Humans; Mass Spectrometry; Melanins; Melanoma, Experimental; Mice; Microphthalmia-Associated Transcription Factor; Plant Extracts; Skin Lightening Preparations; Skin Pigmentation

A green solvent-based optimization for rosmarinic acid (RA), carnosol (COH), and carnosic acid (CA) extraction, the three main antioxidants from rosemary, was performed. The conventional solid-liquid extraction was optimized using a central composite design (CCD) followed by the desirability approach. In the CCD analysis the quantitative effects of extraction time (4.8-55.2min), liquid-to-solid ratio (4.6-21.4mLg(-1)), and ethanol content (44.8-95.2% v/v) were determined for the extracted amount of antioxidants, their concentrations in the extract, and the extraction yield. Samples were analyzed by HPLC and the antioxidants were identified by comparison with pure standard retention times and UV spectra. The desirability function that simultaneously maximizes the antioxidants extraction and their concentrations in the final product was validated. The extraction using a hydroalcoholic solution 70% v/v, at low liquid-to-solid ratio (5mLg(-1)), and after 55-min yielded an antioxidant recovery rate of 89.8%, and a final product 4.75 times richer in the main antioxidants than the raw material.

Topics: Abietanes; Antioxidants; Cinnamates; Depsides; Plant Extracts; Powders; Rosmarinic Acid; Rosmarinus

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

Topics: Abietanes; Anti-Inflammatory Agents; Antineoplastic Agents; Antioxidants; Escherichia coli; Genetic Vectors; Humans; Magnetic Resonance Spectroscopy; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Oxidants; Oxygen; Phylogeny; Rosmarinus; Saccharomyces cerevisiae; Salvia; Spectrometry, Mass, Electrospray Ionization

Topics: Abietanes; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Chromatography, Liquid; Colonic Neoplasms; Gas Chromatography-Mass Spectrometry; HCT116 Cells; HT29 Cells; Humans; Mass Spectrometry; Picrates; Plant Extracts; Rosmarinus

Plant pathogenic fungi are considered of significant economic importance for adversely affecting both quantitatively and qualitatively fresh and processed produce. Extracts of Salvia fruticosa were initially screened for their antifungal activity, and the ethyl acetate fraction, being the most active, was further analyzed using HPLC-SPE-NMR hyphenation. The methoxylated flavones hispidulin, salvigenin, and cirsimaritin and the diterpenes carnosic acid, carnosol, and 12-methoxycarnosic acid were identified as the major components of the extract. In addition, the concentration levels of all identified components were determined using q-NMR. The antifungal activity of the crude extract and selected phytochemicals was estimated against the fungal species Aspergillus tubingensis, Botrytis cinerea, and Penicillium digitatum. The estimated MIC and MFC values of the ethyl acetate extract of S. fruticosa, as well as three of its major constituents, carnosic acid, carnosol, and hispidulin, support their antifungal activity, especially against B. cinerea and P. digitatum, suggesting their potential use in food and agricultural systems.

Topics: Abietanes; Antifungal Agents; Chromatography, High Pressure Liquid; Flavones; Fungi; Magnetic Resonance Spectroscopy; Plant Extracts; Salvia

Carnosic acid (salvin), which possesses antioxidative and antimicrobial properties, is increasingly exploited within the food, nutritional health and cosmetics industries. Since its first extraction from a Salvia species (∼70 years ago) and its identification (∼50 years ago), numerous articles and patents (∼400) have been published on specific food and medicinal applications of Rosmarinus and Salvia plant extracts abundant in carnosic acid. In contrast, relevant biochemical, physiological or molecular studies in planta have remained rare. In this overview, recent advances in understanding of carnosic acid distribution, biosynthesis, accumulation and role in planta, and its applications are summarised. We also discuss the deficiencies in our understanding of the relevant biochemical processes, and suggest the molecular targets of carnosic acid. Finally, future perspectives and studies related to its potential roles are highlighted.

Topics: Abietanes; Animals; Anti-Infective Agents; Antioxidants; Chromatography, High Pressure Liquid; Humans; Mice; Molecular Structure; Plant Leaves; Rats; Rosmarinus; Salvia; Sheep

Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

Topics: Abietanes; Adult; Antioxidants; Apoptosis; Blotting, Western; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cellular Senescence; Female; Fibroblasts; Humans; Immunoenzyme Techniques; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Plant Extracts; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin

To evaluate sodium salicylate (NaSA) as an elicitor of rosmarinic acid (RA) and phenolic diterpenes, carnosol (C) and carnosic acid (CA) production, in a culture of Salvia officinalis shoots.. In sage shoots grown in vitro, 28 polyphenolic compounds (phenolic acids, flavonoids, and phenolic diterpenes) were identified. In shoots treated for 1 week with increasing NaSA concentrations, the content of C increased from 2.3 in control to 5.7 mg g(-1) DW in shoots treated with 500 µM NaSA. In shoots that were recovered on basal medium for 3 weeks, the maximal amount of C (14 mg/g(-1) DW) was with 150 µM NaSA treatment. In treated and recovered shoots, the increase in C was accompanied with a decrease in CA, resulting in 1.9-fold increase in the C/CA ratio. Accumulation of RA was not affected by the NaSA treatment. However, elicitation by NaSA was accompanied with growth retardation.. NaSA can improve C production in sage shoot culture, probably by stimulating the conversion of CA to C.

Topics: Abietanes; Cinnamates; Culture Media; Depsides; Plant Development; Plant Shoots; Rosmarinic Acid; Salvia officinalis; Sodium Salicylate

Induction of phase II enzymes is important in cancer chemoprevention. We compared the effect of rosemary diterpenes on the expression of the pi class of glutathione S-transferase (GSTP) in rat liver Clone 9 cells and the signaling pathways involved. Culturing cells with 1, 5, 10, or 20 μM carnosic acid (CA) or carnosol (CS) for 24 h in a dose-dependent manner increased the GSTP expression. CA was more potent than CS. The RNA level and the enzyme activity of GSTP were also enhanced by CA treatment. Treatment with 10 μM CA highly induced the reporter activity of the enhancer element GPEI. Furthermore, CA markedly increased the translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) from the cytosol to the nucleus after 30 to 60 min. CA the stimulated the protein induction of p38, nuclear Nrf2, and GSTP was diminished in the presence of SB203580 (a p38 inhibitor). In addition, SB203580 pretreatment or silencing of Nrf2 by siRNA suppressed the CA-induced GPEI-DNA binding activity and GSTP protein expression. Knockdown of p38 or Nrf2 by siRNA abolished the activation of p38 and Nrf2 as well as the protein induction and enzyme activity of GSTP by CA. These results suggest that CA up-regulates the expression and enzyme activity of GSTP via the p38/Nrf2/GPEI pathway.

Topics: Abietanes; Active Transport, Cell Nucleus; Animals; Anticarcinogenic Agents; Clone Cells; Electrophoretic Mobility Shift Assay; Enhancer Elements, Genetic; Enzyme Induction; Enzyme Inhibitors; Genes, Reporter; Glutathione S-Transferase pi; Hepatocytes; Imidazoles; Membrane Glycoproteins; NF-E2-Related Factor 2; Pyridines; Rats; Recombinant Proteins; RNA Interference; Rosmarinus; Signal Transduction; Up-Regulation

Abietane diterpenes exhibit an array of interesting biological activities, which have generated significant interest among the pharmacological community. Starting from the abietane diterpenes carnosic acid and carnosol, twenty four new triazole derivatives were synthesized using click chemistry. The compounds differ in the length of the linker and the substituent on the triazole moiety. The compounds were assessed as antiproliferative and antifungal agents. The antiproliferative activity was determined on normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells while the antifungal activity was assessed against Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32264. The carnosic acid γ-lactone derivatives 1-3 were the most active antiproliferative compounds of the series, with IC50 values in the range of 43.4-46.9 μM and 39.2-48.9 μM for MRC-5 and AGS cells, respectively. Regarding antifungal activity, C. neoformans was the most sensitive fungus, with nine compounds inhibiting more than 50% of its fungal growth at concentrations ≤250 µg∙mL-1. Compound 22, possessing a p-Br-benzyl substituent on the triazole ring, showed the best activity (91% growth inhibition) at 250 µg∙mL-1 In turn, six compounds inhibited 50% C. albicans growth at concentrations lower than 250 µg∙mL-1.

Topics: Abietanes; Antifungal Agents; Candida albicans; Cell Line, Tumor; Cell Proliferation; Click Chemistry; Cryptococcus neoformans; Fibroblasts; Humans; Microbial Sensitivity Tests; Neoplasms; Plant Extracts; Structure-Activity Relationship; Triazoles

The antioxidant and antimicrobial effects on lamb meat of the dietary use of rosemary diterpenes and vitamin E were compared. Thirty fattening lambs were assigned to three diets: (C) control; (R) C plus 600 mg kg(-1) carnosic acid and carnosol at 1:1 w:w; or (E) C plus 600 mg kg(-1) α-tocopherol. The deposition of the dietary supplements in the muscle was determined. Microbial quality (total viable counts, Lactic Acid Bacteria, Enterobacteriaceae, Escherichia coli and Salmonella spp), oxidative stability (CIELab color, malondialdehyde and total carbonyls) and sensory attributes (appearance and odor) were determined in loin stored at 2°C under 70% O2/30% CO2 atmosphere. Microbial quality was ensured by packaging and chilling. The E-diet was more effective (P ≤ 0.05) than the R-diet in preventing meat oxidation, although the latter had antimicrobial effects on meat. The shelf life of lamb (assessed as the loss of freshness) could be increased by 5 (R-diet) or 10 (E-diet) days.

Topics: Abietanes; Animals; Anti-Infective Agents; Antioxidants; Bacteria; Color; Dietary Supplements; Female; Food Microbiology; Food Packaging; Food Preservation; Food Storage; Humans; Male; Meat; Muscles; Odorants; Plant Extracts; Rosmarinus; Sheep; Vitamin E

Ultrasound and microwave as green processes are investigated in this study, focusing on the extraction selectivity towards antioxidant extraction from rosemary leaves. Due to its richness in valuable compounds such as rosmarinic, carnosic and ursolic acids, rosemary is a reference matrix for extraction study. In this work, six alternative processes are compared: ultrasound (bath, reactor and probe), microwave (reflux under microwave, microwave under nitrogen pressure and microwave under vapor pressure). The main result of this study is that selective extraction can be achieved according to extraction techniques and therefore to the extraction process.

Topics: Abietanes; Chemical Fractionation; Cinnamates; Depsides; Green Chemistry Technology; Microwaves; Rosmarinic Acid; Rosmarinus; Terpenes; Triterpenes; Ultrasonic Waves; Ursolic Acid

The use of dietary rosemary extract (DRE) at low doses is proposed as a nutritional strategy to improve meat preservation. Lamb diet was supplemented with 0, 200 or 400mg DRE (containing carnosic acid and carnosol at 1:1 w:w) per kg feed during the fattening stage. Meat quality was evaluated in lamb fillets packed under protective atmosphere and kept in retail conditions for up to 14 days. The effects of diet and storage time were determined on different physical-chemical (L*a*b* color, pH, TBARS, protein oxidation and volatiles from lipid oxidation), microbial (total viable and psychrophilic bacteria, Enterobacteriaceae, molds and yeasts) and sensory (appearance and odor) characteristics of the meat. The antioxidant and antimicrobial effects of DRE on meat were demonstrated. DRE delayed lean and fat discoloration, lipid oxidation, odor deterioration and microbial spoilage, extending the shelf life time of fillets from around 9 to 13 days. Both DRE doses provided similar shelf life extension.

Topics: Abietanes; Animal Feed; Animals; Anti-Infective Agents; Antioxidants; Color; Dietary Supplements; Food Microbiology; Food Preservation; Humans; Lipid Peroxidation; Meat; Odorants; Plant Extracts; Rosmarinus; Sheep, Domestic

In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Biological Transport; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glutathione; HT29 Cells; Humans; Inactivation, Metabolic; Metabolomics; Plant Extracts; Polyphenols; Putrescine; Reactive Oxygen Species; Rosmarinus; Signal Transduction; Transcription, Genetic

Ethanol extracts from dried leaves of wild Lepechinia salvia (Lindl) Epling, collected during the flowering period (September-November), contained 15% to 25% carnosic acid and 2 to 8% carnosol, depending on the month of collection. The highest concentration of carnosic acid in extracts was in October, while carnosol concentration had a peak in September, which suggests that it is not a product of carnosic acid oxidation. A comparison of extracts obtained in September 2008 to 2011 shows that the production of both abietanes increased in years with less winter rainfall and higher temperatures, which induced an early blooming. EC50 values in DPPH radical scavenging and antiproliferative (CCRF-CEM tumor cells) bioassays confirm that the high bioactivity of the extracts of rosemary, sage and L. salviae does not arise only from carnosol and carnosic acid. The cytotoxic activity was significantly higher in extracts of L. salviae, probably due to water stress differences between the cultivars and the wild species. These results correlate well with the close phylogenetic relationship between the three species, and their similar medicinal uses.

Topics: Abietanes; Antineoplastic Agents; Antioxidants; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Ethanol; Humans; Lamiaceae; Picrates; Plant Extracts; Plant Leaves

Propionibacterium acnes is a key pathogen involved in the progression of acne inflammation. The development of a new agent possessing antimicrobial and anti-inflammatory activity against P. acnes is therefore of interest. In this study, we investigated the inhibitory effect of rosemary (Rosmarinus officinalis) extract on P. acnes-induced inflammation in vitro and in vivo. The results showed that ethanolic rosemary extract (ERE) significantly suppressed the secretion and mRNA expression of proinflammatory cytokines, including interleukin (IL)-8, IL-1β, and tumor necrosis factor-α in P. acnes-stimulated monocytic THP-1 cells. In an in vivo mouse model, concomitant intradermal injection of ERE attenuated the P. acnes-induced ear swelling and granulomatous inflammation. Since ERE suppressed the P. acnes-induced nuclear factor kappa-B (NF-κB) activation and mRNA expression of Toll-like receptor (TLR) 2, the suppressive effect of ERE might be due, at least partially, to diminished NF-κB activation and TLR2-mediated signaling pathways. Furthermore, three major constituents of ERE, carnosol, carnosic acid, and rosmarinic acid, exerted different immumodulatory activities in vitro. In brief, rosmarinic acid significantly suppressed IL-8 production, while the other two compounds inhibited IL-1β production. Further study is needed to explore the role of bioactive compounds of rosemary in mitigation of P. acnes-induced inflammation.

Topics: Abietanes; Acne Vulgaris; Animals; Anti-Inflammatory Agents; Cinnamates; Cytokines; Depsides; Humans; Inflammation; Inflammation Mediators; Male; Mice; Mice, Inbred ICR; Phytotherapy; Plant Extracts; Propionibacterium acnes; RNA, Messenger; Rosmarinic Acid; Rosmarinus; Signal Transduction

Carnosic acid (CA) and derived diterpenes abundant in rosemary extracts (REs) exert anti-obesity effects. The aim of this study was to investigate the bioavailability of these compounds in a rat model of obesity.. A total of 26 compounds were tentatively identified based on accurate mass information and the isotopic pattern provided by TOF-MS analyzer. The main metabolites detected in the gut content, liver, and plasma were the glucuronide conjugates of CA, carnosol, and rosmanol. Two other metabolites were also identified: CA 12-methyl ether and 5,6,7,10-tetrahydro-7-hydroxyrosmariquinone. All the metabolites were detected as early as 25 min following oral administration. Most of the compounds remained in the intestine, liver, and (or) plasma at substantial concentrations for several hours supporting their potential health benefits in these tissues. We also corroborated the presence of small quantities of CA and detected trace quantities of the main CA metabolites in the brain. Notably, we did not find significant differences in the metabolic profile between lean and obese rats.. We report for the first time a comprehensive profile of metabolites in various organs following the oral consumption of an RE enriched in CA and contribute to establish the potential bioactive molecules.

Topics: Abietanes; Animals; Biological Availability; Brain; Chromatography, Liquid; Diterpenes; Female; Glucuronides; Intestinal Mucosa; Intestines; Liver; Metabolome; Oils, Volatile; Plant Extracts; Rats; Rats, Zucker; Rosmarinus; Tandem Mass Spectrometry

This report describes the development and application of a novel graphene-epoxy composite electrode as a sensitive amperometric detector of capillary electrophoresis. The composite electrode was fabricated on the basis of the in situ polycondensation of a mixture of graphene and 1,2-ethanediamine-containing bisphenol A epoxy resin in the inner bore of a piece of fused silica capillary under heat. The structure of the material was investigated by scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, and Fourier transform infrared spectroscopy. The results indicated that graphene sheets were well dispersed and interconnected throughout the composite to form an electrically conductive network. The performance of this unique electrode was demonstrated by separating and detecting two naturally occurring phenolic compounds in rosemary in combination with capillary electrophoresis. The graphene-based detector offered significantly lower operating potentials, higher sensitivity, satisfactory resistance to surface fouling, and lower expense of operation, indicating great promise for a wide range of applications.

Topics: Abietanes; Electrodes; Electrophoresis, Capillary; Epoxy Resins; Graphite; Models, Chemical; Plant Extracts; Rosmarinus

The use of rosemary (Rosmarinus officinalis) leaves and their constituents as a source of dietary antioxidants and flavoring agents is continuously growing. Carnosol and carnosic acid, two major components of rosemary extracts, have shown activity for cancer prevention and therapy.. In this study, we investigate the cytotoxic and anti-angiogenic activities of carnosol and carnosic acid, in order to get further insight into their mechanism of action.. Our results demonstrate that the mentioned diterpenes inhibit certain functions of endothelial cells, namely, differentiation, proliferation, migration and proteolytic capability. Our data indicate that their growth inhibitory effect, exerted on proliferative endothelial and tumor cells, could be due to, at least in part, an induction of apoptosis. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallantoic membrane assay.. The anti-angiogenic activity of carnosol and carnosic acid could contribute to the chemopreventive, antitumoral and antimetastatic activities of rosemary extracts and suggests their potential in the treatment of other angiogenesis-related malignancies.

Topics: Abietanes; Angiogenesis Inhibitors; Animals; Antioxidants; Apoptosis; Cattle; Cell Movement; Cells, Cultured; Collagen; Drug Combinations; HL-60 Cells; Human Umbilical Vein Endothelial Cells; Humans; Laminin; Matrix Metalloproteinase 2; Plant Extracts; Plant Leaves; Proteoglycans; Rosmarinus; Vascular Endothelial Growth Factor Receptor-2

Carnosic acid, a diterpene in rosemary, is considered to be beneficial in the prevention of chronic neurodegenerative diseases. Recently, it has been found that drugs with antiangiogenic activity lower the risk of neurodegenerative diseases. Thus it is of interest whether carnosic acid has antiangiogenic activity. In this study, carnosic acid suppressed microvessel outgrowth on ex vivo angiogenesis assay using a rat aortic ring at higher than 10 µM. The antiangiogenic effect of carnosic acid was found in angiogenesis models using human umbilical vein endothelial cells with regard to tube formation on reconstituted basement membrane, chemotaxis and proliferation. Although the carnosol in rosemary also suppressed angiogenesis, its effect was not more potent than that of carnosic acid in the ex vivo model. These results suggest that carnosic acid and rosemary extract can be useful in the prevention of disorders due to angiogenesis, and that their antiangiogenic effect can contribute to a neuroprotective effect.

Topics: Abietanes; Angiogenesis Inhibitors; Animals; Aorta; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Male; Neovascularization, Physiologic; Neuroprotective Agents; Plant Extracts; Plant Leaves; Rats; Rats, Wistar; Rosmarinus

Rosemary (Rosmarinus officinalis L.) is a popular culinary/medicinal herb. Recent studies have shown it has pharmacologic activities for cancer chemoprevention and therapy. This study evaluated the antiproliferation activity of rosemary extract (RE) against human ovarian cancer cells, and whether the extract and its three main active ingredients carnosol (CS), carnosic acid (CA) and rosmarinic acid (RA) can enhance the antiproliferation activity of cisplatin (CDDP). Our study showed that RE has significant antiproliferation activity on human ovarian cancer A2780 and its CDDP resistant daughter cell line A2780CP70, with IC(50) (50% inhibitory concentration) estimated at 1/1000 and 1/400 dilutions respectively. RE enhanced the antiproliferation effect with CDDP on both A2780 and A2780CP70 cells. A2780 cells were consistently more sensitive to CS, CA, and RA than A2780CP70 cells between 2.5 and 20μg/ml. CS and RA also showed synergistic antiproliferation effect with CDDP on A2780 cells at some concentrations. RE treated by ultrafiltration, dialysis, and removal of phenolics lost the antiproliferation activity suggested that the activity resides in phenolics with MW<1000Da. Apoptosis array study of A2780 cells treated with RE showed that the expression of a number of genes regulating apoptosis were modulated by the treatment. This study showed that RE inhibited the proliferation of ovarian cancer cell lines by affecting the cell cycle at multiple phases. It induced apoptosis by modifying the expression of multiple genes regulating apoptosis, and holds potential as an adjunct to cancer chemotherapy.

Topics: Abietanes; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cinnamates; Cisplatin; Depsides; Drug Therapy, Combination; Female; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; Plant Extracts; Plant Leaves; Rosmarinic Acid; Rosmarinus

Prostaglandin E(2) (PGE(2)), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE(2) synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE(2) in a cell-free assay by direct interference with microsomal PGE(2) synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC(50) values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC(50) values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE(2) generation upon stimulation with lipopolysaccharide (IC(50) = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF(1α), 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B(2)] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE(2) formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE(2) formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

Topics: Abietanes; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Humans; Intramolecular Oxidoreductases; Microsomes; Plant Extracts; Plant Leaves; Prostaglandin-E Synthases; Prostaglandins; Salvia officinalis

Rosemary, whose major caffeoyl-derived and diterpenoid ingredients are rosmarinic acid, carnosol, and carnosic acid, is an important source of natural antioxidants and is being recognized increasingly as a useful preservative, protectant, and even as a potential medicinal agent. Understanding the stability of these components and their mode of interaction in mixtures is important if they are to be utilized to greatest effect. A study of the degradation of rosmarinic acid, carnosol, carnosic acid, and a mixture of the three was conducted in ethanolic solutions at different temperatures and light exposure. As expected, degradation increased with temperature. Some unique degradation products were formed with exposure to light. Several degradation products were reported for the first time. The degradation products were identified by HPLC/MS/MS, UV, and NMR. The degradation of rosemary extract in fish oil also was investigated, and much slower rates of degradation were observed for carnosic acid. In the mixture of the three antioxidants, carnosic acid serves to maintain levels of carnosol, though it does so at least in part at the cost of its own degradation.

Topics: Abietanes; Antioxidants; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Kinetics; Plant Extracts; Rosmarinic Acid; Rosmarinus; Temperature

The importance of the diterpenic and rosmarinic acid content in the biological activities of rosemary extracts has been studied previously, but how the relationship between the concentration of these components affects their antioxidant and antibacterial activities has received little attention. Accordingly, from a total of 150 plants, 27 methanolic extracts were selected, for their similar diterpene contents but different ratios between carnosic acid and carnosol concentrations. In extracts with similar rosmarinic acid contents but differing proportions between carnosic acid and carnosol, the two diterpenes were seen to equally affect the in vitro antioxidant activity; however, and related with the antibacterial efficiency, this biological activity improved when carnosol was the major diterpene component.

Topics: Abietanes; Anti-Bacterial Agents; Antioxidants; Cinnamates; Depsides; Listeria monocytogenes; Methanol; Plant Extracts; Rosmarinic Acid; Rosmarinus; Staphylococcus aureus

Atherosclerosis is a chronic and progressive inflammatory disease. Novel anti-inflammatory therapies may have promise as treatment strategies for cardiovascular risk reduction. Rosemary (Rosemarinus officinalis L.) has been used in folk medicine to treat headaches, epilepsy, poor circulation, and many other ailments. It was found that rosemary could act as a stimulant and mild analgesic and could reduce inflammation. However, the mechanisms underlying the anti-inflammatory and antiatherosclerotic effects of rosemary need more study. This study investigated effects of the rosemary components, carnosic acid (CA), and carnosol (CAR), on cell migration. Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) were determined by Western blot and gelatin zymography, respectively, in RAW 264.7 macrophages and vascular smooth muscle cells (VSMCs). VSMC migration was assessed by a Matrigel migration assay. Active compounds of rosemary extracts were also analyzed using a reversed-phase high-performance liquid chromatography. MMP-9 and MCP-1 activities were markedly diminished with methanol extract (RM), n-hexane fraction (RH), and CA in RAW 264.7 cells. RM, RH, CA, and CAR suppressed tumor necrosis factor-alpha-induced VSMC migration by inhibiting MMP-9 expression. Chromatograms of RM- and RH-containing CA and CAR revealed higher CA contents of RM (9.4%, 93.85 μg/mg dry wt.) and, especially, RH (18.4%, 184.00 μg/mg dry wt.), which were appreciably elevated compared with the similar CAR content in RM and RH (3.7%, 37.30 μg/mg dry wt.; and 2.5%, 25.05 μg/mg dry wt., respectively). Rosemary, especially its CA component, has potential antiatherosclerosis effects related to cell migration.

Topics: Abietanes; Animals; Anti-Inflammatory Agents; Atherosclerosis; Blotting, Western; Cell Line; Cell Movement; Chemokine CCL2; Chromatography, High Pressure Liquid; Matrix Metalloproteinase 9; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Plant Extracts; Rosmarinus; Tumor Necrosis Factor-alpha

Polyphenol antioxidants decrease the risk of atherosclerosis. The study aimed to evaluate prospectively in healthy young participants the effect of oral rosemary extracts (RE), consisting of diphenols, upon endothelial dysfunction (ED), preceding structural atherosclerosis. Nineteen healthy young volunteers were studied prospectively, who received oral RE (77.7 mg) for 21 days, consisting of active substances carnosol (0.97 mg), carnosic (8.60 mg) and rosmarinic acid (10.30 mg). Before and after RE treatment, the study evaluated fasting serum levels of plasminogen-activator-inhibitor-1 (PAI-1), vascular cell adhesion molecule 1 (VCAM-1), inter-cellular adhesion molecule 1 (ICAM-1), superoxide dismutase (SOD), glutathione peroxidase (GPX), fibrinogen, high-sensitivity capsular reactive protein (hs-CRP), tumor-necrosis factor α (TNF-α), the lipid profile and ED, characterized as flow-mediated dilatation (FMD) in the brachial artery of < 4.5%, estimated by ultrasound measurements. After 21 days, any side effects were registered, the mean FMD increased nonsignificantly (6.51 ± 5.96% vs 7.78 ± 4.56%, p = 0.546) and ED decreased significantly (66.6% vs 16.6%, p = 0.040). Among the serum markers, only the mean PAI-1 level decreased significantly (4.25 ± 1.46 U/mL vs 3.0 ± 0.61 U/mL, p = 0.012) after 21-day RE supplementation. It is concluded that oral RE supplementation has the potential to improve serum PAI-1 activity and ED in young and healthy individuals.

Topics: Abietanes; Administration, Oral; Adult; Brachial Artery; Cinnamates; Depsides; Endothelium, Vascular; Female; Humans; Male; Plant Extracts; Plasminogen Activator Inhibitor 1; Prospective Studies; Rosmarinic Acid; Rosmarinus; Ultrasonography; Vasodilation; Vasodilator Agents

The anticarcinogenic effect of rosemary has been partly attributed to the modulation of the activity and expression of phase II detoxification enzymes. Here we compared the effects of phenolic diterpenes from rosemary on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Cells were treated with 1-20 μmol/L of carnosic acid (CA) or carnosol (CS) for 24 h. Both CA and CS dose dependently increased NQO1 enzyme activity and protein expression, and the induction potency of CA was stronger than that of CS. The increase in NQO1 enzyme activity in cells treated with 10 μmol/L CA and CS was 4.1- and 1.9-fold, respectively (P < 0.05). RT-PCR showed that CA and CS induced NQO1 mRNA in a dose-dependent manner. Furthermore, CA dose dependently induced transcription of nuclear factor erythroid-2 related factor 2 (Nrf2) and antioxidant response element (ARE)-luciferase reporter activity. Silencing of Nrf2 expression alleviated NQO1 protein expression and ARE-luciferase activity by CA. Moreover, the phosphorylation of p38 was mainly stimulated in the presence of CA. Pretreatment with SB203580 or silencing of p38 expression inhibited Nrf2 activation and NQO1 induction. These results suggest that the increased NQO1 expression by CA is likely related to the p38-Nrf2 pathway and help to clarify the possible molecular mechanism of action of rosemary phenolic compounds in drug metabolism and cancer prevention.

Topics: Abietanes; Animals; Anticarcinogenic Agents; Antioxidants; Blotting, Western; Cell Nucleus; Cells, Cultured; Dose-Response Relationship, Drug; Gene Silencing; Liver; Luciferases; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Plasmids; Rats; Response Elements; RNA, Messenger; Signal Transduction; Transcription, Genetic; Transfection; Up-Regulation

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.

Topics: Abietanes; Animals; Antioxidants; Cell Line; Drug Evaluation, Preclinical; Gene Expression Profiling; Glutathione; Mice; Neuroprotective Agents; Oligonucleotide Array Sequence Analysis; Plant Extracts; Rosmarinus

Carnosic acid (2), a major anti-oxidant in rosemary (Rosmarinus officinalis), was synthesized from pisiferic acid (1), the major constituent of Sawara (Chamaecyparis pisifera), via ortho-oxidation of the phenol using meta-chlorobenzoyl peroxide (mCBPO), chloroacetyl meta-chlorobenzoyl peroxide (CAMCBPO) or 2-iodoxybenzoic acid (IBX). Carnosol (3), another anti-oxidant in rosemary, was synthesized from carnosic acid by oxidation with silver oxide. Potent antibacterial activities against Propionibacterium acnes (ATCC 6919) (minimum inhibitory concentration (MIC) mug/ml) and Staphylococcus aureus ME/GM/TC Resistant (ATCC 33592) (MIC mug/ml) of carnosic acid and carnosol were reported.

Topics: Abietanes; Anti-Bacterial Agents; Antioxidants; Chamaecyparis; Diterpenes; Plant Extracts; Propionibacterium acnes; Rosmarinus; Staphylococcus aureus

The in vitro inhibitory activity of crude EtOH/H2O extracts from the leaves and stems of Rosmarinus officinalis L. was evaluated against the following microorganisms responsible for initiating dental caries: Streptococcus mutans, S. salivarius, S. sobrinus, S. mitis, S. sanguinis, and Enterococcus faecalis. Minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The bioassay-guided fractionation of the leaf extract, which displayed the higher antibacterial activity than the stem extract, led to the identification of carnosic acid (2) and carnosol (3) as the major compounds in the fraction displaying the highest activity, as identified by HPLC analysis. Rosmarinic acid (1), detected in another fraction, did not display any activity against the selected microorganisms. HPLC Analysis revealed the presence of low amounts of ursolic acid (4) and oleanolic acid (5) in the obtained fractions. The results suggest that the antimicrobial activity of the extract from the leaves of R. officinalis may be ascribed mainly to the action of 2 and 3.

Topics: Abietanes; Anti-Infective Agents; Dental Caries; Enterococcus faecalis; Microbial Sensitivity Tests; Plant Extracts; Plant Leaves; Plant Stems; Rosmarinus; Streptococcus

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.

Topics: 3T3-L1 Cells; Abietanes; Active Transport, Cell Nucleus; Adipocytes; Adipogenesis; Animals; Enzymes; G2 Phase; Gene Expression; Glutathione; Mice; NF-E2-Related Factor 2; Oligonucleotide Array Sequence Analysis; Plant Extracts; Response Elements

Carnosic acid (CA) and carnosol (CS) are phenolic diterpenes present in several labiate herbs like Rosmarinus officinalis (Rosemary) and Salvia officinalis (Sage). Extracts of these plants exhibit anti-inflammatory properties, but the underlying mechanisms are largely undefined. Recently, we found that CA and CS activate the peroxisome proliferator-activated receptor gamma, implying an anti-inflammatory potential on the level of gene regulation. Here we address short-term effects of CA and CS on typical functions of human polymorphonuclear leukocytes (PMNL). We found that (I), CA and CS inhibit the formation of pro-inflammatory leukotrienes in intact PMNL (IC(50)=15-20 microM [CA] and 7 microM [CS], respectively) as well as purified recombinant 5-lipoxygenase (EC number 1.13.11.34, IC(50)=1 microM [CA] and 0.1 microM [CS], respectively), (II) both CA and CS potently antagonise intracellular Ca(2+) mobilisation induced by a chemotactic stimulus, and (III) CA and CS attenuate formation of reactive oxygen species and the secretion of human leukocyte elastase (EC number 3.4.21.37). Together, our findings provide a pharmacological basis for the anti-inflammatory properties reported for CS- and CA-containing extracts.

Topics: Abietanes; Calcium; Homeostasis; Humans; Lipoxygenase Inhibitors; Neutrophils; Plant Extracts; Reactive Oxygen Species

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.

Topics: Abietanes; Adaptor Proteins, Signal Transducing; Animals; Antioxidants; Carboxylic Acids; Catechols; Cell Line; Cytoprotection; Cytoskeletal Proteins; Drug Evaluation, Preclinical; Esterases; Gene Expression Regulation; Glutathione; Hydroxyl Radical; Kelch-Like ECH-Associated Protein 1; Membrane Lipids; Mice; Molecular Structure; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Phenanthrenes; Plant Extracts; Response Elements; Steroids

The effects of 24 h supplementation of Caco-2 cells with carnosic acid and carnosol, and their activities against 5 microM oleic acid hydroperoxide (OAHPx)-mediated oxidative stress, were investigated. At 24 h of incubation, under nonstressed and stressed conditions, both compounds at 25, 50, and 100 microM supplement concentrations reduced catalase activity, whereas changes in glutathione peroxidase and superoxide dismutase activities varied depending upon the concentrations. Relative to control cultures, carnosic acid and carnosol reduced membrane damage by 40-50% when stressed by OAHPx. Carnosic acid and carnosol inhibited lipid peroxidation by 88-100% and 38-89%, respectively, under oxidative stress conditions. Both compounds significantly lowered DNA damage induced by OAHPx. Results of this study suggest that antioxidant activities of carnosic acid and carnosol could be partly due to their ability to increase or maintain glutathione peroxidase and superoxide dismutase activities.

Topics: Abietanes; Antioxidants; Caco-2 Cells; Humans; Lipid Peroxides; Oxidative Stress; Phenanthrenes; Plant Extracts; Rosmarinus

We found that a crude extract from Salvia officinalis (sage) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). We isolated the effective compound from the extract and identified it as carnosol, one of diterpenoids. Carnosol showed a weak antimicrobial activity, and greatly reduced the MICs of various aminoglycosides (potentiated the antimicrobial activity of aminoglycosides) and some other types of antimicrobial agents in VRE. Carnosic acid, a related compound, showed the similar activity. The effect of carnosol and carnosic acid with gentamicin was synergistic.

Topics: Abietanes; Aminoglycosides; Anti-Bacterial Agents; Drug Synergism; Enterococcus faecalis; Enterococcus faecium; Methicillin Resistance; Microbial Sensitivity Tests; Phenanthrenes; Plant Extracts; Plant Leaves; Pseudomonas aeruginosa; Salvia officinalis; Serratia marcescens; Staphylococcus aureus; Vancomycin Resistance

Carnosol and carnosic acid, two antioxidant polyphenols present in Rosmarinus officinalis (rosemary), were investigated for their antiproliferative properties toward Caco-2 cells. Twenty hours of treatment with both carnosol and carnosic acid inhibited 3H-thymidine incorporation in a dose-dependent manner, with a 50% inhibitory concentration of 23 microM and significantly increased the doubling time of Caco-2 cells from 29.5 to 140 and 120 h, respectively. These effects were associated with accumulation of treated cells in the G2/M phase of the cell cycle. Carnosol was found to exert its major cell cycle effect after prometaphase, and caused an increase in cyclin B1 protein levels whereas carnosic acid arrested cells prior to prometaphase, and caused a reduction in cyclin A levels. These structurally related phytochemicals, therefore, appear to arrest cells at different phases of the cell cycle possibly through influencing the levels of different cyclin proteins.

Topics: Abietanes; Antineoplastic Agents; Antioxidants; Caco-2 Cells; Cell Division; Cell Proliferation; Cyclin A; Cyclin B; Cyclin B1; Down-Regulation; G2 Phase; Humans; Inhibitory Concentration 50; Phenanthrenes; Plant Extracts

Rosmarinus officinalis extracts were investigated by a combination of bioassays and biochemical analysis to identify bioactive compounds. The 2,2-diphenyl-2-picrylhydracyl hydrate (DPPH) radical scavenging method, Folin-Ciocaulteau method and HPLC chromatography were used to study the distribution and levels of antioxidants (AOXs). Antimicrobial activity analysis was carried out using the disk diffusion and broth dilution techniques. A good correlation between the AOX activities and total phenol content in the extracts was found. Although all rosemary extracts showed a high radical scavenging activity, a different efficacy as antimicrobial agent was observed. Methanol extract containing 30% of carnosic acid, 16% of carnosol and 5% of rosmarinic acid was the most effective antimicrobial against Gram positive bacteria (minimal inhibition concentration, MIC, between 2 and 15 mug/ml), Gram negative bacteria (MIC between 2 and 60 mug/ml) and yeast (MIC of 4 mug/ml). By contrast, water extract containing only 15% of rosmarinic acid showed a narrow activity. MIC value of the methanol and water extracts is in a good correlation with the values obtained with pure carnosic acid and rosmarinic acid, respectively. Therefore, our results suggested that the antimicrobial rosemary extracts efficacy was associated with their specific phenolic composition. Carnosic acid and rosmarinic acid may be the main bioactive antimicrobial compounds present in rosemary extracts. From a practical point of view, rosemary extract may be a good candidate for functional foods as well as for pharmaceutical plant-based products.

Topics: Abietanes; Anti-Infective Agents; Antioxidants; Bacteria; Biphenyl Compounds; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Flavonoids; Free Radicals; Indicators and Reagents; Phenanthrenes; Phenols; Picrates; Plant Extracts; Polyphenols; Rosmarinic Acid; Rosmarinus; Saccharomyces cerevisiae

The radioprotective effects of carnosic acid (CA), carnosol (COL), and rosmarinic acid (RO) against chromosomal damage induced by gamma-rays, compared with those of L-ascorbic acid (AA) and the S-containing compound dimethyl sulfoxide (DMSO), were determined by use of the micronucleus test for antimutagenic activity, evaluating the reduction in the frequency of micronuclei (MN) in cytokinesis-blocked cells of human lymphocytes before and after gamma-ray irradiation. With treatment before gamma-irradiation, the most effective compounds were, in order, CA > RO > or = COL > AA > DMSO. The radioprotective effects (antimutagenic) with treatment after gamma-irradiation were lower, and the most effective compounds were CA and COL. RO and AA presented small radioprotective activity, and the sulfur-containing compound DMSO lacked gamma-ray radioprotection capacity. Therefore, CA and COL are the only compounds that showed a significant antimutagenic activity both before and after gamma-irradiation treatments. These results are closely related to those reported by other authors on the antioxidant activity of the same compounds, and the degree of effectiveness depends on their structure. Furthermore, the results for treatments before and after gamma-ray irradiation suggest the existence of different radioprotective mechanisms in each case.

Topics: Abietanes; Antimutagenic Agents; Cell Nucleus; Cinnamates; Depsides; DNA Damage; Gamma Rays; Lymphocytes; Phenanthrenes; Plant Extracts; Radiation-Protective Agents; Rosmarinic Acid; Rosmarinus

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand activated transcription factor, belonging to the metazoan family of nuclear hormone receptors. Activation of PPARgamma increases the transcription of enzymes involved in primary metabolism, leading to lower blood levels of fatty acids and glucose. Hence, PPARgamma represents the major target for the glitazone type of drugs currently being used clinically for the treatment of type 2 diabetes. Furthermore, activators of PPARgamma show beneficial anti-inflammatory and anti-tumour effects. Utilizing a fusion receptor of the yeast Gal4-DNA binding domain joined to the hinge region and ligand binding domain of the human PPARgamma in combination with a Gal4-driven luciferase reporter gene, cotransfected into Cos7 cells, we tested sage and rosemary extracts prepared with 80 % aqueous ethanol for possible PPARgamma activation. This revealed that both extracts are capable of selectively activating Gal4-PPARgamma fusion receptor, in a concentration-dependent manner, with EC (50) values of 22.8 +/- 8.4 mg/L and 33.7 +/- 7.3 mg/L for rosemary and sage, respectively. Subsequent analysis of the characteristic constituents revealed the phenolic diterpene compounds carnosol, present in both herbs, and carnosic acid to be active principles of these extracts, showing EC (50) values of 41.2 +/- 5.9 microM and 19.6 +/- 2.0 microM, respectively. Thus it can be concluded that the glucose lowering effect reported recently for rosemary may be attributed to PPARgamma activation. Moreover, our observations may also explain the anti-inflammatory and antiproliferative effects of both compounds published previously.

Topics: Abietanes; Animals; Chlorocebus aethiops; COS Cells; Genes, Reporter; Humans; Phenanthrenes; Plant Extracts; PPAR gamma; Recombinant Fusion Proteins; Rosmarinus; Salvia officinalis

As part of a project to characterise plant-derived natural products that modulate bacterial multidrug resistance (MDR), bioassay-guided fractionation of a chloroform extract of the aerial parts of Rosmarinus officinalis led to the characterisation of the known abietane diterpenes carnosic acid (1), carnosol (2) and 12-methoxy-trans-carnosic acid. Additionally, a new diterpene, the cis A/B ring junction isomer of 12-methoxy-trans-carnosic acid, 12-methoxy-cis-carnosic acid (5), was isolated. The major components were assessed for their antibacterial activities against strains of Staphylococcus aureus possessing efflux mechanisms of resistance. Minimum inhibitory concentrations ranged from 16 to 64 microg/ml. Incorporation of 1 and 2 into the growth medium at 10 microg/ml caused a 32- and 16-fold potentiation of the activity of erythromycin against an erythromycin effluxing strain, respectively. Compound 1 was evaluated against a strain of S. aureus possessing the NorA multidrug efflux pump and was shown to inhibit ethidium bromide efflux with an IC50 of 50 microM, but this activity is likely to be related to the inhibition of a pump(s) other than NorA. The antibacterial and efflux inhibitory activities of these natural products make them interesting potential targets for synthesis.

Topics: Abietanes; Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Phenanthrenes; Plant Extracts; Rosmarinus; Staphylococcus aureus

Subcritical water extraction at several temperatures ranging from 25 to 200 degrees C has been studied to selectively extract antioxidant compounds from rosemary leaves. An exhaustive characterization of the fractions obtained using subcritical water at different temperatures has been carried out by LC-MS, and the antioxidant activities of the extracts have been measured by a free radical method (DPPH). Results indicate high selectivity of the subcritical water toward the most active compounds of rosemary such as carnosol, rosmanol, carnosic acid, methyl carnosate, and some flavonoids such as cirsimaritin and genkwanin. The antioxidant activity of the fractions obtained by extraction at different water temperatures was very high, with values around 11.3 microg/mL, comparable to those achieved by SFE of rosemary leaves. A study of the effect of the temperature on the extraction efficiency of the most typical rosemary antioxidant compounds has been performed.

Topics: Abietanes; Antioxidants; Chromatography, High Pressure Liquid; Chromatography, Liquid; Diterpenes; Flavonoids; Mass Spectrometry; Molecular Structure; Phenanthrenes; Plant Extracts; Plant Leaves; Rosmarinus; Temperature; Water

The distribution of six compounds with three different polyphenol skeletons have been studied in Rosmarinus officinalis: phenolic diterpenes (carnosic acid, carnosol, and 12-O-methylcarnosic acid), caffeoyl derivatives (rosmarinic acid), and flavones (isoscutellarein 7-O-glucoside and genkwanin), each showing a characteristic behavior and distribution during the vegetative cycle. Only in leaves were all six compounds present, and the highest accumulation rate was related with the young stages of development. Rosmarinic acid showed the highest concentrations of all the polyphenols in all organs. The distribution of this acid in leaves, flowers, and stems suggests that in the first stages of flower growth, levels were due to in situ biosynthesis, and in the last stages, the contribution of transport phenomena was increased. The antioxidant activity of six extracts with different polyphenolic composition was evaluated in aqueous and lipid systems. The results clearly suggest that rosemary extracts are excellent antioxidants in both aqueous and lipid systems.

Topics: Abietanes; Antioxidants; Cinnamates; Depsides; Diterpenes; Flavonoids; Flowers; Phenanthrenes; Phenols; Plant Extracts; Plant Leaves; Plant Roots; Plant Stems; Polymers; Rosmarinic Acid; Rosmarinus

The antioxidant activity of aqueous infusions of sage emerges from specific components present in that herb. In an attempt to investigate the chemical nature and properties of these components, four organic solvent extracts from aqueous infusions of sage were examined. HPLC analyses of these extracts led to the separation of a number of components, of which four were identified and quantified through the use of standard compounds of known chromatographic HPLC profiles. These compounds are the diterpenes carnosic acid, carnosol, and rosmanol and the hydroxycinnamic acid caffeic acid. The antioxidant activity and polyphenol content were determined in the four organic solvent extracts and the left-over aqueous fraction. Both polyphenolic and nonpolyphenolic substances present in the extracts arise as significant contributors to the observed antioxidant activity of the derived extracts and thus sage itself. In this sense, they reflect the antioxidant potential of the aqueous infusions of sage toward reactive oxygen species generated through variable mechanisms of iron-promoted oxidative processes.

Topics: Abietanes; Antioxidants; Caffeic Acids; Chromatography, High Pressure Liquid; Diterpenes; Flavonoids; Phenanthrenes; Phenols; Plant Extracts; Polyphenols; Salvia officinalis; Water

Preparative-scale incubations with Nocardia sp. NRRL 5646 were conducted to produce new derivatives of the abietane diterpene chemoprotectant and antioxidant carnosic acid (1). Reductive biotransformation of the C-20 carboxylic acid functional group followed by biological methylation at the C-11 phenol afforded 4. Oxidative cyclization of 1 to carnosol 5 followed by dihydroxylation at the isopropyl moiety afforded 6. Metabolites 4 and 6 are new carnosic acid derivatives whose structures were confirmed by mass spectrometry and NMR spectroscopic analysis. The radical quenching properties of 4-6 using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging assay showed activities similar to that of mixed tocopherols and carnosic acid.

Topics: Abietanes; Antioxidants; Biphenyl Compounds; Cyclization; Diterpenes; Free Radical Scavengers; Mass Spectrometry; Methylation; Molecular Structure; Nocardia; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Picrates; Plant Extracts; Rosmarinus; Structure-Activity Relationship

The known diterpenes rosmanol (3), rosmaquinone (4), 7-methoxyrosmanol (5), 7-ethoxyrosmanol (6), galdosol (7), and epirosmanol (8) have been obtained by partial synthesis from carnosol (2), an abundant natural product present in Salvia species. The physical and spectroscopic data of these semisynthetic diterpenes were identical to those of authentic natural samples and with data reported in the literature. These abietane diterpenes have very interesting biological activities and are present in the genus Salviain low quantities; thus, the semisynthetic approach described here represents an efficient alternative method to obtain these compounds. Additionally, the known diterpene 16-hydroxyrosmanol (10) and a new aromatic diterpene 11 were obtained from 16-hydroxycarnosol (9) by reaction with Ph3P/NBS in CH2Cl2. The structure of the new compound 11 was established from its spectroscopic data as 12,16-epoxycarnosol.

Topics: Abietanes; Catalysis; Diterpenes; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Phenanthrenes; Plant Extracts; Plant Leaves; Plants, Medicinal; Salvia

Carnosol 1, rosmanol 2, carnosic acid 3 and 20-deoxocarnosol 4, the main phenolic abietanes present in aerial parts of the Chilean medicinal plant Sphacele salviae were tested for antioxidant activity of measuring the decay of the radical cation diphenyl-picrylhydrazyl (DPPH). All compounds displayed higher antioxidant levels than BHT under the same conditions and carnosic acid was more efficient than vitamin E.

Topics: Abietanes; Antioxidants; Biphenyl Compounds; Butylated Hydroxytoluene; Chile; Diterpenes; Dose-Response Relationship, Drug; Lamiaceae; Medicine, Traditional; Phenanthrenes; Picrates; Plant Extracts; Plant Leaves; Plants, Medicinal; Structure-Activity Relationship; Time Factors; Vitamin E

Oxidative stress is implicated in the etiology of cancer, hence compounds that alleviate oxidative stress by inducing enzymes that defend against free radical damage might be useful as cancer chemopreventives. Glutathione S-transferase (GST) has been suggested to be a candidate for a critical enzyme in protecting cells against free radical damage, in part, because its level of induction correlates with protection of the cell line IMR-32 against hydrogen peroxide-induced oxidative stress. The present study identified dietary ortho phenols that both induce GST and protect the cell line IMR-32 against hydrogen peroxide-caused oxidative stress. The ortho phenol (o-phenol) inducers were better protectors against oxidative stress than a number of GST inducers that did not bear phenolic groups, possibly because the phenol residues of the ortho phenols allowed their action as antioxidants as well as inducers of GST. GST has previously been thought to protect cells against cancer by detoxifying mutagenic xenobiotics. The present results suggest that ortho phenol inducers of GST might be useful as cancer chemopreventives that act by two independent mechanisms, the alleviation of oxidative stress and the detoxification of mutagenic xenobiotics.

Topics: Abietanes; Anticarcinogenic Agents; Antioxidants; Diet; Diterpenes; Enzyme Induction; Flavonoids; Flavonols; Glutathione Transferase; Humans; Hydrogen Peroxide; Inactivation, Metabolic; Inhibitory Concentration 50; Isomerism; Mutagens; Oxidative Stress; Phenanthrenes; Phenols; Plant Extracts; Structure-Activity Relationship; Tumor Cells, Cultured

Abietane diterpenes were measured in field-grown rosemary (Rosmarinus officinalis L.) plants throughout the year. Carnosic acid and carnosol, which were present in high amounts (5 and 1 mg/gDW respectively) in rosemary leaves, decreased by ca. 50% during the Mediterranean summer in response to low precipitation, high radiation and high temperature. In contrast, the highly oxidised diterpenes rosmanol, isorosmanol and dimethyl isorosmanol, which are formed from carnosic acid by enzymatic dehydrogenation and the action of activated oxygen, increased in response to such environmental constraints. Collectively, these data support the contention that abietane diterpenes from R. officinalis function as antioxidants that protect biological membranes from oxidative stress. This is especially important during the Mediterranean summer when there is low precipitation, high light and high temperature.

Topics: Abietanes; Antioxidants; Climate; Diterpenes; Lamiaceae; Mediterranean Region; Oxidative Stress; Phenanthrenes; Plant Extracts

Extracts of herbs and spices are increasingly of interest in the food industry because they retard oxidative degradation of lipids. There is also increasing interest in the antiviral activity of plant products. A liquid, deodorized rosemary extract and an oily extract of a mixture of Provençal herbs were tested for antioxidant and antiviral action in vitro. The rosemary extract (Herbor 025) and the extract of Provençal herbs (Spice Cocktail) inhibited peroxidation of phospholipid liposomes with 50% inhibition concentration values of 0.0009% (v/v) and 0.0035% (v/v), respectively. Herbor 025 and the spice cocktail (at 0.2%, v/v) reacted with trichloromethylperoxyl radical with calculated rates of 2.7 x 10(4) s-1 and 1.5 x 10(3) s-1, respectively. The main active components in the herbal preparations, carnosol and carnosic acid, at 0.05% (v/v) react with rate constants of (1-3) x 10(6) M-1 sec-1 and 2.7 x 10(7) M-1 sec-1, respectively. Both extracts show good antioxidant activity in the Rancimat test, especially in lard. Herbor 025 and the spice cocktail inhibited human immunodeficiency virus (HIV) infection at very low concentrations which were also cytotoxic. However, purified carnosol exhibited definite anti-HIV activity at a concentration (8 microM) which was not cytotoxic. Both preparations promoted some DNA damage in the copper-phenanthroline and the bleomycin-iron systems. The two herbal preparations possess antioxidant properties that may make them useful in the food matrix.

Topics: Abietanes; Antioxidants; Antiviral Agents; Biological Assay; Diterpenes; DNA Damage; Evaluation Studies as Topic; HIV-1; Humans; Lipid Peroxidation; Liposomes; Lymphoma, T-Cell; Magnoliopsida; Phenanthrenes; Plant Extracts; Spices; T-Lymphocytes; Tumor Cells, Cultured

Carnosic acid, an antioxidant extracted from rosemary, is shown to produce radicals when in contact with oxidized methyl oleate in the absence of air above 50 degrees C. Two radical species are formed: the first one, stable up to approximately 110 degrees C, is an hydroxy-phenoxy radical whose ESR spectrum was analyzed by studying its temperature dependence and its sensitivity to deuterium/proton exchange. The second species was observed above 110 degrees C, its ESR spectrum was identical to the spectrum obtained when carnosol, another antioxidant extracted from rosemary, was heated at the same temperature in the presence of oxidized lipid. This observation is probably due to the transformation of carnosic acid into carnosol; the analysis of the corresponding ESR spectrum suggests the formation of a keto phenoxy radical exhibiting a great delocalization of the unpaired electron.

Topics: Abietanes; Antioxidants; Diterpenes; Electron Spin Resonance Spectroscopy; Free Radicals; Lipids; Oleic Acids; Phenanthrenes; Plant Extracts; Spices

1. Carnosol and carnosic acid have been suggested to account for over 90% of the antioxidant properties of rosemary extract. 2. Purified carnosol and carnosic acid are powerful inhibitors of lipid peroxidation in microsomal and liposomal systems, more effective than propyl gallate. 3. Carnosol and carnosic acid are good scavengers of peroxyl radicals (CCl3O2.) generated by pulse radiolysis, with calculated rate constants of 1-3 x 10(6) M-1 s-1 and 2.7 x 10(7) M-1 s-1 respectively. 4. Carnosic acid reacted with HOCl in such a way as to protect the protein alpha 1-antiproteinase against inactivation. 5. Both carnosol and carnosic acid stimulated DNA damage in the bleomycin assay but they scavenged hydroxyl radicals in the deoxyribose assay. The calculated rate constants for reaction with .OH in the deoxyribose system for carnosol and carnosic acid were 8.7 x 10(10) M-1 s-1 and 5.9 x 10(10) M-1 s-1 respectively. 6. Carnosic acid appears to scavenge H2O2, but it could also act as a substrate for the peroxidase system. 7. Carnosic acid and carnosol reduce cytochrome c but with a rate constant significantly lower than that of O2(-.).

Topics: Abietanes; Antioxidants; Bleomycin; Carnosine; Deoxyribose; Diterpenes; DNA Damage; Free Radical Scavengers; Free Radicals; Horseradish Peroxidase; Hydrogen Peroxide; Hypochlorous Acid; Lipid Peroxidation; Magnoliopsida; Oxidants; Phenanthrenes; Plant Extracts; Superoxides

The phenolic diterpene carnosic acid appears to be the main substance for general oxidation leading to artifacts with gamma- or delta-lactone structure in extracts of Rosmarinus officinalis and Salvia officinalis. Until now it was only possible to prepare carnosic acid by hydrogenolysis of carnosol. A semipreparative HPLC method has been developed isolating carnosic acid among other phenolic diterpenes. The separated substances were identified by 13C-nuclear magnetic resonance (NMR), 1H-NMR, mass and IR spectroscopy. Conversion of carnosic acid and carnosol to other phenolic diterpenes was investigated by HPLC.

Topics: Abietanes; Antioxidants; Carnosine; Chromatography, High Pressure Liquid; Diterpenes; Magnetic Resonance Spectroscopy; Magnoliopsida; Mass Spectrometry; Oxidation-Reduction; Phenanthrenes; Plant Extracts; Spectrophotometry, Infrared