Recovery mechanism of the antioxidant activity from carnosic acid quinone,an oxidized sage and rosemary antioxidant

A solution of carnosic acid quinone, which is a radical chain-termination product having no antioxidant activity in the antioxidant reaction of carnosic acid, recovers potent antioxidant activity upon standing. The HPLC analysis of an aged solution of carnosic acid quinone revealed that several antioxidants are produced in the solution. From the time-course and quantitative analyses of the formation of the products and their structural analysis, an antioxidant mechanism from carnosic acid quinone is proposed that includes a redox reaction of carnosic acid quinone in addition to the isomerization to lactone derivatives. In the first stage of antioxidation, carnosic acid, the reduction product from carnosic acid quinone, contributes to the potent antioxidant activity of the solution. This proposed mechanism can explain one of the reasons for the strong antioxidant activity of the extract of the popular herbs sage and rosemary.     

Subcritical water extraction of antioxidant compounds from rosemary plants

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.     

Degradation Study of Carnosic Acid, Carnosol, Rosmarinic Acid, and Rosemary Extract (Rosmarinus officinalis L.) Assessed Using HPLC

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.     

Enhanced carnosic acid levels in two rosemary accessions exposed to cold stress conditions

Two rosemary accessions were subjected to chilling temperatures in control environmental cabins analyzing their variations in rosmarinic and carnosic acids together with their adaptability to these stress conditions. Cold stressed plants of both accessions showed increases in caffeic acid and carnosic acid concentration levels, while other secondary metabolites such as rosmarinic acid, naringin, cirsimaritin, hispidulin, and carnosol showed different responses in both accessions. In addition, cold stressed plants exhibited significant reductions in chlorophylls, beta-carotene, and violaxanthin levels as well as the maximum quantum yield of PSII in both accessions. Hydrogen peroxide and lipid peroxidation levels showed similar responses in both accessions, which were positively and negatively correlated with rosmarinic and carnosic acids. From these results it is therefore suggested that carnosic acid biosynthesis in rosemary plants is induced by chilling periods. On the other hand, we demonstrate that not all rosemary accessions are equally well adapted to chilling temperatures. In fact, for (one) accession cold treated plants severe losses in chlorophyll, beta-carotene, and even xanthophylls (including zeaxanthin and antheraxanthin) were observed, despite no visual symptoms of leaf injury. More research is needed to understand rosmarinic acid variations in rosemary plants under stress conditions.     

Antioxidant mechanism of carnosic acid: structural identification of two oxidation products.

To determine the antioxidant mechanism of food phenolics against the oxidation of food components, the reaction of carnosic acid, an antioxidative constituent of the popular herbs sage and rosemary, was investigated in the presence of ethyl linoleate and the radical oxidation initiator 2,2'-azobis(2,4-dimethylvaleronitrile). During this process, carnosic acid was oxidized to an o-quinone and a hydroxy p-quinone, the chemical structures of which were confirmed by physical and chemical techniques. From a quantitative time course analysis of the production of these quinones, an antioxidant mechanism of carnosic acid is proposed, consisting of the oxidative coupling reaction with the peroxyl radical at the 12- or 14-position of carnosic acid and subsequent degradation reactions.     

Low-density lipoprotein, collagen, and thrombin models reveal that Rosemarinus officinalis L. exhibits potent antiglycative effects

Using the low-density lipoprotein (LDL), collagen, and thrombin models, we report here that the rosemary extracts (REs), either the aqueous (REw) or the acetonic (REA), all possessed many antiglycation-related features, and the effective concentrations required were as follows: 0.1 mg/mL for suppressing the relative electrophoretic mobility, 1.3 microg/mL for anticonjugated diene induction, 0.5 mg/mL for inhibition of thiobarbituric acid reactive substances production, 0.1 mg/mL for AGEs (advanced glycation end products) formation, 0.1 mg/mL to block glucose incorporation, and 0.05 mg/mL as an effective anti-antithrombin III. Using high-performance liquid chromatography/mass spectrometry, we identified five major constituents among eight major peaks, including rosmarinic acid, carnosol, 12-methoxycarnosic acid, carnosic acid, and methyl carnosate. In the LDL model, REA was proven to be more efficient than REw; yet, the reverse is true for the collagen and the thrombin III models, the reason of which was ascribed to the higher lipid-soluble antioxidant content (such as rosmarinic acid, carnosol, carnosic acid, 12-methoxycarnosic acid and methyl carnosate) in REA than in REw and the different surface lipid characteristics between LDL and collagen; although to act as anti-AGEs, both extracts were comparable. To assist the evidence, a larger 2,2-diphenyl-1-picrylhydrazyl radical scavenging capability with less total polyphenolic content was found in REA. We conclude that rosemary is an excellent multifunctional therapeutic herb; by looking at its potential potent antiglycative bioactivity, it may become a good adjuvant medicine for the prevention and treatment of diabetic, cardiovascular, and other neurodegenerative diseases.     

Polyphenolic transmission to Segureno lamb meat from ewes' diet supplemented with the distillate from rosemary (Rosmarinus officinalis) leaves

The aim of the present work is to study whether the introduction of rosemary plant byproduct, from plant steam distillation, in daily Segurena sheep feeding allows the transfer of active antioxidant components to lamb meat, without detriment to the animal productivity. For this, 36 Segurena ewes were assigned randomly to three homogeneous groups. One group was fed a basal diet as a control and the diet of the other two groups was modified by substituting 10 or 20% of the control diet (respectively) with distilled rosemary leaves. Chromatographic analysis allowed the identification of 11 polyphenolic components previously identified in the rosemary and basal diet pellets, respectively. Among them, rosmarinic acid, carnosol, and carnosic acid were the phenolic components that had a significantly increased presence ( P < 0.05) in the lamb meat from sheep mothers fed this aromatic herb, when compared to the control group. The incorporation of this byproduct into the animal diet favored the antioxidant capacity of these lamb meat samples. Fresh meat produced on rosemary had higher total ferric reducing antioxidant power (FRAP) ( P < 0.05), greater ability to reduce ABTS*+, and lower IC50 (DPPH*) ( P < 0.05) values when compared to the control group. Because no statistically significant differences were detected among the results obtained from the lamb meat belonging to the different ewe groups fed rosemary leaf extract (10 or 20%), it can be concluded that the incorporation of distilled rosemary leaves at a rate of 10% of the ewes' diet should be enough to improve the lamb meat antioxidant status.     

Phenolic diterpenes,flavones, and rosmarinic acid distribution during the development of leaves,flowers, stems,and roots of Rosmarinus officinalis. Antioxidant activity

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.     

Relevance of carnosic acid concentrations to the selection of rosemary, Rosmarinus officinalis (L.), accessions for optimization of antioxidant yield

Methods were developed to identify and select accessions of rosemary, Rosmarinus officinalis (L.), producing optimum antioxidant activity. Extracts from 12 different rosemary accessions, using three solvents of varying polarity, were assayed for their antioxidant activity, and their major antioxidant compounds were identified and quantified by high-performance liquid chromatography (HPLC). Carnosic acid concentrations were correlated with (i) the free radical scavenging activity of these extracts, as measured by the 2,2-diphenyl-1-picrylhydrazyl assay (adjusted R(2) = 77.3%) and (ii) their inhibition of linoleic acid oxidation, as measured by the beta-carotene assay (adjusted R(2) = 44.1%). The correlation was broadly confirmed by the production of volatile aldehydes as measured by the hexanal assay. The variation of carnosic acid concentrations in extracts of 29 accessions, grown in field trials at three sites in England, was determined.     

Anti-inflammatory effects of supercritical carbon dioxide extract and its isolated carnosic acid from Rosmarinus officinalis leaves

Rosemary (Rosmarinus officinalis) leaves possess a variety of bioactivities. Previous studies have shown that the extract of rosemary leaves from supercritical fluid extraction inhibits the expression of inflammatory mediators with apparent dose-dependent responses. In this study, three different extraction conditions (5000 psi at 40, 60, and 80 °C) of supercritical carbon dioxide (SC-CO(2)) toward the extraction of antioxidants from rosemary were investigated. Furthermore, simultaneous comparison of the anti-inflammatory properties between rosemary extract prepared from SC-CO(2) under optimal conditions (5,000 psi and 80 °C) and its purified carnosic acid (CA) using lipopolysaccharide (LPS)-treated murine RAW 264.7 macrophage cells was also presented. Results showed that the yield of 3.92% and total phenolics of 213.5 mg/g extract obtained from the most effective extraction conditions showed a high inhibitory effect on lipid peroxidation (IC(50) 33.4 μg/mL). Both the SC-CO(2) extract and CA markedly suppressed the LPS-induced production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), as well as the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated inhibitor-kappaB (P-IκB), and nuclear factor-kappaB (NF-κB)/p65 in a dose-dependent manner. The five major compounds of verbenone, cirsimaritin, salvigenin, carnosol, and CA existing in the SC-CO(2) extract were isolated by semipreparative HPLC and identified by HPLC-MS/MS analysis. CA was the most abundant recorded compound and the most important photochemical with an anti-inflammatory effect with an IC(50) of 22.5 μM or 7.47 μg/mL presented to the best inhibitory activity on NO production better than that of the 14.50 μg/mL dosage prepared from the SC-CO(2) extract. Nevertheless, the effective inhibition of LPS-induced NF-κB signaling in RAW 264.7 cells from the SC-CO(2) extract extends the potential application of nutraceutical formulation for the prevention of inflammatory diseases.     

Phytochemical stability and color retention of copigmented and processed muscadine grape juice

Muscadine (Vitis rotundifolia) grape juice was assessed for color and phytochemical stability as influenced by anthocyanin copigmentation with a water-soluble rosemary extract, fortification with ascorbic acid, and processing by heat or high hydrostatic pressure (HHP). The roles of polyphenolic cofactors in the presence and in the absence of ascorbic acid were assessed as a means to improve the overall processing stability of the juice. Addition of rosemary extract from 0 to 0.4% (v/v) readily formed copigment complexes with anthocyanins and resulted in concentration-dependent hyperchromic shifts from 10 to 27% that corresponded to increased antioxidant activity. The presence of ascorbic acid was generally detrimental to juice quality, especially in the presence of rosemary extract, and resulted in overall anthocyanin, ascorbic acid, and antioxidant activity losses. Although thermal and high-pressure processing methods were detrimental to juice quality, HHP resulted in greater losses after processing, likely due to action from residual oxidase enzymes. Although physicochemical attributes were enhanced by copigmentation with rosemary extract, methods to inactivate residual enzymes should be addressed prior to copigmentation to prevent degradation of anthocyanins in the presence of ascorbic acid.     

Characterization of two unknown compounds in methanol extracts of rosemary oil

In this study, two unknown compounds in rosemary oil, containing 3% carnosic acid and 0.3% carnosol, were identified and characterized. After methanol extraction, purification, and analysis by reversed-phase HPLC and LC-MS, a recovery of 92% (+/-8%) of carnosic acid was obtained, but no carnosol was found. However, two unknown compounds with a molecular weight of 330.2 and 302.2 were consistently detected. From additional LC-MS-MS, (1)H NMR, and elemental analyses, it became clear that the first compound (M(w) = 330.2) could not be carnosol. It was hypothesized that it originated from the breakdown of the intramolecular bond of carnosol, followed by the addition of a water molecule. Possibly, an unsaturated double bond was formed after dehydration. Assuming that this compound was an intermediate in the conversion to rosmanol, the second unknown compound (M(w) = 302.2) may have resulted from the breakdown of the intramolecular bond of rosmanol. Similarly, an unsaturated double bond may have been formed. After splitting off carbon oxide, a detectable molecule with a molecular weight of 302.2 was observed.     

Ionizing radiation and antioxidants affect volatile sulfur compounds, lipid oxidation, and color of ready-to-eat Turkey bologna

Bologna was processed from ground turkey breast meats containing one of four antioxidant treatments (none, rosemary extract, sodium erythorbate, and sodium nitrite). After it was cooked, the bologna was sliced, sealed in gas impermeable bags, exposed to 0, 1.5, and 3.0 kGy gamma-radiation, and then stored at 5 degrees C for up to 8 weeks. Thiobarbuturic acid reactive substances (TBARS), color, and volatile sulfur compounds were measured every 2 weeks during storage. Irradiation had no consistent effect on TBARS values. The rosemary extract and sodium nitrite inhibited, while erythorbate increased, TBARS values, independent of radiation dose or storage time. Irradiation promoted redness and reduced yellowness of the control (no antioxidant) bologna at weeks 0 and 2. The use of nitrite and rosemary extract inhibited the changes in color due to irradiation. Several volatile sulfur compounds (hydrogen sulfide, methanethiol, methyl sulfide, and dimethyl disulfide), measured using a pulsed flame photometric detector, increased with radiation dose. However, none of the antioxidants had any substantial effect on volatile sulfur compounds induced by irradiation. Our results suggest that antioxidants did not consistently affect irradiation-induced volatile sulfur compounds of turkey bologna although they did significantly impact color and lipid oxidation.     

Antioxidant activity of a catechol derived from abietic acid

The demand for novel effective antioxidant-based drugs has led to the synthesis and evaluation of the antioxidant potential in several molecules derived from natural compounds. In this work the in vitro antioxidant activity of an abietic acid-derived catechol (methyl 11,12-dihydroxyabietate-8,11,13-trien-18-oate, MDTO) was evaluated. This substance, possessing important biological properties, is similar to carnosic acid, a natural antioxidant from rosemary or sage leaves. Aiming to understand the antioxidant activity of MDTO, the energetics of its O-H bond, using time-resolved photoacoustic calorimetry (TR-PAC), was investigated. On energetic grounds it is predicted that MDTO is a good free radical scavenger, although its activity is lower than that of quercetin, a very effective antioxidant, which was used for comparison. In agreement with these predictions, the DPPH(*) and ABTS(*+) radical scavenging activities are lower than those of quercetin. In addition, MDTO also reacts with HOCl, a powerful proinflammatory oxidant produced by activated neutrophils, and protects liposomes against iron-ascorbate-induced oxidation. The discussion of these results foresees potential applications of MDTO as an antioxidant.     

Enhancement of polyphenol recovery from rosemary (Rosmarinus officinalis) and sage (Salvia officinalis) by enzyme-assisted ensiling (ENLAC).

The efficacy of enzyme-assisted ensiling (ENLAC) in the recovery of polyphenols from rosemary and sage was tested. Fresh rosemary and sage were chopped and ensiled in 0.5-L anaerobic jars. Treatments comprised control (no additives), 0.5% glucose and lactic acid bacteria, and 1% cellulase plus 1% hemicellulase plus pectinase. Following storage at room temperature for 45 days (experiment 1) and 26 days (experiment 2), polyphenols were extracted from the silages in ethanol either by direct blending or by cold extraction. The enzyme treatment resulted in silages with the lowest pH values, lowest fiber content, highest water-soluble sugar content, and highest polyphenol recovery; this treatment resulted in increased polyphenol recovery from rosemary and sage, by 100 and 20%, respectively. Comparison between direct blending and cold extraction revealed similar efficiency of polyphenol recovery.     

Effects of olive, canola, and sunflower oils on the formation of volatiles from the Maillard reaction of lysine with xylose and glucose

Some important edible oils (extra virgin olive oil, canola oil, and sunflower oil) were added to aqueous glucose-lysine or xylose-lysine model systems to investigate their effect on the formation of volatiles from the Maillard reaction (MR). The volatile compounds were extracted by a Likens-Nickerson apparatus and quantified. Pyrazines, Maillard reaction products with an important impact on food flavor, appeared to be particularly sensitive to the presence of the oils in both the xylose-lysine and glucose-lysine model systems. The unsubstituted pyrazine was formed more with olive oil, less with canola oil, and even less with sunflower oil, whereas 2-methylpyrazine, 2,5-methylpyrazine, and 2,3-dimethylpyrazine were formed less with olive oil, more with canola oil, and even more with sunflower. The oxidative states of the oils and their fatty acid fingerprints were determined: the results indicated that the relative amounts of the pyrazines are sensitive to the degree of unsaturation of the oil. The autoxidation of the volatile compounds generated from the MR, investigated by the addition of free radical modulators (antioxidants alpha-tocopherol, 2,6-di-tert-butyl-4-methylphenol, and rosemary extract; or pro-oxidant alpha,alpha'-azobis-isobutyronitrile, a free radical initiator), was limited in respect to aqueous model systems.     

Autoxidation in xylose/lysine model systems

The volatiles produced in xylose/lysine model systems added with an antioxidant (alpha-tocopherol, 2,6-di-tert-butyl-4-methylphenol, or rosemary extract) or a free radical initiator (alpha, alpha'-azobis(isobutyronitrile), AIBN) were analyzed to investigate the effects of the presence of free radicals on the Maillard reaction. The pH was maintained constant at 4 or 6, by adding a base, and the data were compared by principal component analysis (PCA). The additives were more effective at pH 4 than pH 6. At pH 4, the model system added with AIBN is very well-discriminated by PCA from the models with the antioxidants and the reference model system, indicating that the volatiles are sensitive to compounds that can interfere in an opposite way with free radical formation.     

Ionic interactions and salinity affect monoterpene and phenolic diterpene composition in rosemary (Rosmarinus officinalis)

In this study, we evaluated how increased cation supply can alleviate the toxic effects of NaCl on plants and how it affects essential oils (EOs) and phenolic diterpene composition in leaves of rosemary (Rosmarinus officinalis L.) plants grown in pots. Two concentrations of the chloride salts KCl, CaCl₂, MgCl₂, and FeCl₃ were used together with 100 mM NaCl to study the effects of these nutrients on plant mineral nutrition and leaf monoterpene, phenolic diterpene, and EO composition. The addition of 100 mM NaCl, which decreased K⁺, Ca²⁺, and Mg²⁺ concentrations with increasing Na⁺ in leaves, significantly altered secondary metabolite accumulation. Addition of MgCl₂ and FeCl₃ altered leaf EO composition in 100 mM NaCl–treated rosemary plants while KCl and CaCl₂ did not. Furthermore, addition of CaCl₂ promoted the accumulation of the major phenolic diterpene, carnosic acid, in the leaves. The carnosol concentration was reduced by the addition of KCl to salt‐stressed plants. It is concluded that different salt applications in combination with NaCl treatment may have a pronounced effect on phenolic diterpene and EO composition in rosemary leaves thus indicating that ionic interactions may be carefully considered in the cultivation of these species to achieve the desired concentrations of these secondary metabolites.     

Foaming properties of wheat gliadin

We studied gliadin solubility, surface tension and foam behavior, and the presence of different gliadin types in gliadin aqueous solutions and foams as a function of pH. Gliadin has excellent foaming properties only at neutral and alkaline pH. Its solubility is minimal near neutral pH, while almost complete at acidic and alkaline pH. Surface tensions of gliadin solutions are minimal around neutral pH, higher at alkaline pH, and highest at acidic pH, which corresponds well with their respective foaming properties. Foams at acidic and alkaline pH values are enriched in γ-gliadin, while foams at pH 8.0 have a similar distribution of α- and γ-gliadins. Thus, γ-gliadin predominantly contributes to the foaming properties of gliadin. The poor foaming properties of gliadin at pH 2.0 improve in the presence of 0.25 and 1.0% NaCl. It follows that the presence of positively charged amino acid residues hinders the formation of stable foam at acidic pH.