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Although used in in vitro culture to boost secondary metabolite production, UV-B radiation can seriously affect plant growth if not properly dosed. Rosemary callus can be used as an important source of effective ingredients in the food and medicine industry. To balance the positive and negative effects of UV-B on rosmary callus, this study investigated the effects of melatonin on rosemary callus under UV-B radiation. The results showed that melatonin improved rosemary callus growth, with fresh weight and dry weight increased by 15.81% and 8.30%, respectively. The addition of 100 μM melatonin increased antioxidant enzyme activity and NO content in rosemary callus. At the same time, melatonin also significantly reduced membrane lipid damage and H 2 O 2 accumulation in rosemary callus under UV-B stress, with malondialdehyde (MDA) and H 2 O 2 contents reduced by 13.03% and 14.55%, respectively. In addition, melatonin increased the total phenol and rosmarinic acid contents in rosemary callus by 19% and 54%, respectively. Melatonin significantly improved the antioxidant activity of the extracts from rosemary callus. These results suggest that exogenous melatonin can alleviate the adverse effects of UV-B stress on rosemary callus by promoting NO accumulation while further enhancing phenolic accumulation and biological activity., (© 2024 Scandinavian Plant Physiology Society.)

High-quality genome of rosemary (Salvia rosmarinus) represents a valuable resource and tool for understanding genome evolution and environmental adaptation as well as its genetic improvement. However, the existing rosemary genome did not provide insights into the relationship between antioxidant components and environmental adaptability. In this study, by employing Nanopore sequencing and Hi-C technologies, a total of 1.17 Gb (97.96%) genome sequences were mapped to 12 chromosomes with 46 121 protein-coding genes and 1265 non-coding RNA genes. Comparative genome analysis reveals that rosemary had a closely genetic relationship with Salvia splendens and Salvia miltiorrhiza, and it diverged from them approximately 33.7 million years ago (MYA), and one whole-genome duplication occurred around 28.3 MYA in rosemary genome. Among all identified rosemary genes, 1918 gene families were expanded, 35 of which are involved in the biosynthesis of antioxidant components. These expanded gene families enhance the ability of rosemary adaptation to adverse environments. Multi-omics (integrated transcriptome and metabolome) analysis showed the tissue-specific distribution of antioxidant components related to environmental adaptation. During the drought, heat and salt stress treatments, 36 genes in the biosynthesis pathways of carnosic acid, rosmarinic acid and flavonoids were up-regulated, illustrating the important role of these antioxidant components in responding to abiotic stresses by adjusting ROS homeostasis. Moreover, cooperating with the photosynthesis, substance and energy metabolism, protein and ion balance, the collaborative system maintained cell stability and improved the ability of rosemary against harsh environment. This study provides a genomic data platform for gene discovery and precision breeding in rosemary. Our results also provide new insights into the adaptive evolution of rosemary and the contribution of antioxidant components in resistance to harsh environments., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The present study aimed to assess the impact of grape seed extract (GSE), onion peel extract (OPE), and rosemary extract (ROE) on Diquat-induced growth restriction and oxidative stress in Lohmann chicks. A total of 200 chicks were randomly assigned to 5 diets: the positive control (PC) group, the negative control (NC) group, GSE group, OPE group, and ROE group. During the first 7 d of trial, compared with NC and PC groups, the GSE group enhanced average daily feed intake (ADFI). From day 8-21, diquat injection resulted in reduced growth performance, increased platelet volume distribution width (PWD), malondialdehyde (MDA) concentration, and activities of alanine aminotransferase (ALT) in chick serum; it also decreased total protein (TP), albumin (ALB), globulin (GLB) concentration, activities of superoxide dismutase (SOD) and glutathione S-transferase (GST) in chick serum; furthermore, it increased MDA concentration while decreasing GST activities in liver. The NC group exhibited lower average daily gain (ADG) than other groups. Compared with NC group, GSE group reduced ALT activities, MDA levels, and red cell distribution width (RDW), and PDW concentration; it also increased SOD, GST activities. The ROE group lowered ALT activities and MDA concentration. The OPE group decreased ALT activities, and MDA levels, RDW, and PDW concentration, and increased SOD activities of chicks. These results suggest that supplementing antioxidants in diets alleviated oxidative stress in chicks challenged by improving antioxidant capacity and liver function.

Melatonin is a stress-related hormone that plays a critical role in triggering the plant defence system and regulating secondary metabolism when plants are exposed to stress. To explore the potential roles of melatonin in response to Ultraviolet-B (UV-B) radiation, we examined the effects of exogenous melatonin on rosemary in vitro shoots under UV-B stress. The application of melatonin (50 μM) alleviated the adverse effects of UV-B stress on the biomass, photosynthetic pigment contents, and membrane lipids of the rosemary in vitro shoots. Melatonin significantly increased superoxide dismutase (1.15.1.1, SOD), peroxidase (1.11.1.7, POD), and catalase (1.11.1.6, CAT) activities by 62%, 99%, and 53%, respectively. The contents of total phenols, rosmarinic acid, and carnosic acid increased under UV-B stress, and they further increased by the melatonin treatment by 41%, 68%, and 67%, respectively, compared with the control group. Under UV-B stress, the increased total phenol content in melatonin-pretreated plants could be attributed to the activation of phenylalanine ammonia-lyase (4.3.1.5, PAL) and tyrosine aminotransferase (2.6.1.5, TAT). In addition, melatonin enhanced the antioxidant and antibacterial activities of the rosemary in vitro shoots under UV-B stress. These results suggest that melatonin can alleviate the damage caused by UV-B stress and also enhance the secondary metabolism and bioactivity of rosemary in vitro shoots., (© 2023 Scandinavian Plant Physiology Society.)

Rosemary essential oil (REO) has been used for several medical purposes. Previous studies have shown the antinociceptive effect of the oil. This study aimed to investigate the role of some well-known receptors in the antinociceptive effect of REO. Male Swiss mice (25-30 g) were used. To assess the antinociceptive activity, the formalin test was used. At first, the antinociceptive effect of three doses of rosemary oil (150, 300 and 450 µL/kg) was tested, and then a dose of 300 µL/kg was selected for the mechanistic study. Animals were pretreated with several antagonists and enzyme inhibitors to evaluate the role of adrenergic, cholinergic, serotoninergic, dopaminergic and opioid receptors as well as the NO/cGMP/K ATP pathway in the antinociceptive effect of rosemary essential oil. Yohimbine (5 mg/kg), prazocin (2 mg/kg), propranolol (2 mg/kg), atropine (2.5 mg/kg) naloxone (5 mg/kg), cyproheptadine (2 mg/kg), ondansetron (2 mg/kg) and haloperidol (1 mg/kg) could not reverse the antinociceptive effect. Sulpiride (20 mg/kg) only showed preventive activity in the early phase of formalin test while methylene blue (5 mg/kg), L-NAME (20 mg/kg) and glibenclamide (10 mg/kg) significantly attenuated the antinociceptive effect of REO in both phases. Tadalafil (2 mg/kg) potentiated the antinociceptive effect of REO in the late phase of formalin test and arginine (100 mg/kg) had no effect on both phases. Therefore the NO/cGMP/K ATP pathway might have an important role in the antinociceptive effect of REO., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11 Gb in size; the genome has a scaffold N50 value of 95.5 Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Alzheimer's disease (AD) has been associated with the hallmark features of cholinergic dysfunction, amyloid beta (Aβ) aggregation and impaired synaptic transmission, which makes the associated proteins, such as β-site amyloid precursor protein cleaving enzyme 1 (BACE I), acetylcholine esterase (AChE) and synapsin I, II and III, major targets for therapeutic intervention. The present study investigated the therapeutic potential of three major phytochemicals of Rosmarinus officinalis, ursolic acid (UA), rosmarinic acid (RA) and carnosic acid (CA), based on their binding affinity with AD-associated proteins. Detailed docking studies were conducted using AutoDock vina followed by molecular dynamic (MD) simulations using Amber 20. The docking analysis of the selected molecules showed the binding energies of their interaction with the target proteins, while MD simulations comprising root mean square deviation (RMSD), root mean square fluctuation (RMSF) and molecular mechanics/generalized born surface area (MM/GBSA) binding free energy calculations were carried out to check the stability of bound complexes. The drug likeness and the pharmacokinetic properties of the selected molecules were also checked through the Lipinski filter and ADMETSAR analysis. All these bioactive compounds demonstrated strong binding affinity with AChE, BACE1 and synapsin I, II and III. The results showed UA and RA to be potential inhibitors of AChE and BACE1, exhibiting binding energies comparable to those of donepezil, used as a positive control. The drug likeness and pharmacokinetic properties of these compounds also demonstrated drug-like characteristics, indicating the need for further in vitro and in vivo investigations to ascertain their therapeutic potential for AD.

Recent research on familial dysautonomia (FD) has focused on the development of therapeutics that facilitate the production of the correctly spliced, exon 20-containing, transcript in cells and individuals bearing the splice-altering, FD-causing mutation in the elongator acetyltransferase complex subunit I (ELP1) gene. We report here the ability of carnosol, a diterpene present in plant species of the Lamiaceae family, including rosemary, to enhance the cellular presence of the correctly spliced ELP1 transcript in FD patient-derived fibroblasts by upregulating transcription of the ELP1 gene and correcting the aberrant splicing of the ELP1 transcript. Carnosol treatment also elevates the level of the RNA binding motif protein 24 (RBM24) and RNA binding motif protein 38 (RBM38) proteins, two multifunctional RNA-binding proteins. Transfection-mediated expression of either of these RNA binding motif (RBMs) facilitates the inclusion of exon 20 sequence into the transcript generated from a minigene-bearing ELP1 genomic sequence containing the FD-causing mutation. Suppression of the carnosol-mediated induction of either of these RBMs, using targeting siRNAs, limited the carnosol-mediated inclusion of the ELP1 exon 20 sequence. Carnosol treatment of FD patient peripheral blood mononuclear cells facilitates the inclusion of exon 20 into the ELP1 transcript. The increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the sirtuin 2 (SIRT2) transcript, a sentinel for exon 20 inclusion in the FD-derived ELP1 transcript, are observed in RNA isolated from whole blood of healthy adults following the ingestion of carnosol-containing rosemary extract. These findings and the excellent safety profile of rosemary together justify an expedited clinical study of the impact of carnosol on the FD patient population., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Non small cell lung cancer (NSCLC) is a global, fatal oncological malady to which conventional and targeted therapies proved less effective with consequent side effects; hence, phytocomponents from herbal sources may provide potent alternative and should be tested for cancer intervention. Activation and overexpression of proto-oncogene tyrosine kinase Src (c-Src) and focal adhesion kinase (FAK) lead to cell proliferation and invasion. Hence, in the present investigation, in silico analysis was carried out to identify molecular intervention of phytocomponents in blocking the active site and thus inhibiting c-Src and FAK activation, which in turn could control progression of NSCLC., Materials and Methods: In silico analysis was carried out using Molegro Virtual Docker, Molegro Molecular Viewer, and ClusPro server for ligand-protein and protein-protein interaction study. Phytochemical analysis and assay for antioxidant activity of hydroalcoholic extract of Rosmarinus officinalis L. were carried out using standard phytochemical tests, high-performance thin-layer chromatography, and 2, 2-diphenyl-1-picrylhydrazyl assay. Effectiveness of extract in arresting cell proliferation was confirmed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay on A549 cell line., Results: In silico analysis indicated effective binding of rosmarinic acid to the active site of target proteins FAK and c-Src, blocking their activity. MTT assay revealed potent antiproliferative activity of hydroalcoholic extract which acted in dose-dependent manner. Phytochemical analysis confirmed that the extract was rich in phytocomponents and had antioxidant activity of 94.9%, which could therefore effectively eliminate free radicals and inhibit cell progression., Conclusion: In silico and in vitro studies confirmed that phytocomponents present in hydroalcoholic extract of R. officinalis L. could effectively block the active site of target proteins and thus controlled cell proliferation on NSCLC cells, suggesting herb as an effective alternative medicine for the treatment of NSCLC., Competing Interests: None

Background: FOXD1 expression in oral squamous cell carcinoma remains uncovered. The aim was to detect the anticancer effect of Rosemary Extract RE through the evaluation of FOXD1 gene expression in (OSCC) by quantitative PCR., Methods: OSCC cell line was served as a control group. Moreover, the OSCC cell line (SCC-15) was treated with RE (OSCC/ RE group) at 24, 48, and 72 hs time intervals. We assessed the antioxidant activity of RE by evaluation of lipid peroxidation (MDA) and superoxide dismutase (SOD) levels. The cytotoxic effects of RE were examined by MTT assay. mTOR and LC3 I/II autophagy protein markers were assessed by western blot. Apoptosis activity was assessed., Results: The study results were statistically assessed. Intergroup comparisons were analyzed, whereas intragroup comparisons were conducted utilizing one-way repeated measures ANOVA, followed by multiple pairwise paired t-tests with Bonferroni correction revealed a significant increase of FOXD1 gene expression in the control OSCC group in comparison to the OSCC/RE group (p-value <0.001). A significant decrease of mTOR/LC3I/II proteins expression in the OSCC/RE group compared to the control OSCC group (p-value <0.001)., Conclusion: FOXD1 can be considred a diagnostic biomarker for OSCC. RE inhibits autophagy of oral human cancer cells via mTOR/LC3I/II-dependent pathways and decrease caspase -3 apoptotic level.

The present work was showed to assess the effect of administration of rosemary extract on etoposide-induced toxicity, injury and proliferation in male rats were investigated. Forty male albino rats were arranged into four equal groups. 1st group, control; 2nd group, etoposide; 3rd group, co-treated rosemary & etoposide; 4th group, rosemary alone. In comparison to the control group, etoposide administration resulted in a significant increase in serum ALT, AST, ALP, total bilirubin, total protein, and gamma GT. In contrast; a significant decrease in albumin level in etoposide group as compared to G1. G3 revealed a significant decrease in AST, ALT, ALP, total protein and total bilirubin levels and a significant rise in albumin level when compared with G2. Serum levels of urea, creatinine, potassium ions, and chloride ions significantly increased; while sodium ions were significantly decreased in G2 when compared with G1. Also, there was an increase of MDA level for etoposide treated group with corresponding control rats. However, there was a remarkable significant decrease in SOD, GPX and CAT levels in G2 as compared to G1. There was a significant increase in serum hydrogen peroxide (H2O2) and Nitric oxide (NO) levels in group treated with etoposide when compared to control group. It was noticeable that administrated by rosemary alone either with etoposide had not any effect on the levels of H2O2 and Nitric oxide. Serum level of T3 and T4 was significantly increased in etoposide-administered rats in comparison with G1. The administration of rosemary, either alone or with etoposide, increased the serum levels of T3 and T4 significantly when compared to control rats. The gene expression analysis showed significant downregulation of hepatic SOD and GPx in (G2) when compared with (G1). The treatment with rosemary extract produced significant upregulation of the antioxidant enzymes mRNA SOD and GPx. MDA gene was increased in (G2) when contrasted with (G1). Treatment of the etoposide- induced rats with rosemary extract delivered significant decrease in MDA gene expression when compared with etoposide group. Rats treated with etoposide showed significant decline in hepatic Nrf2 protein expression, when compared with G1. While, supplementation of Etoposide- administered rats with the rosemary produced a significant elevation in hepatic Nrf2 protein levels. Additionally, the liver histological structure displayed noticeable degeneration and cellular infiltration in liver cells. It is possible to infer that rosemary has a potential role and that it should be researched as a natural component for etoposide-induced toxicity protection.

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [ 3 H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7- O -methyl- epi -rosmanol, 4',7- O -dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.

To study the effects of Methyl jasmonates (MeJA) on rosemary suspension cells, the antioxidant enzymes' change of activities under different concentrations of MeJA, including 0 (CK), 10 (M10), 50 (M50) and 100 μM MeJA (M100). The results demonstrated that MeJA treatments increased the activities of phenylalanine ammonla-lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and polyphenol oxidase (PPO) and reduced the contents of hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA), thus accelerating the ROS scavenging. Comparative transcriptome analysis of different concentrations of MeJA showed that a total of 7836, 6797 and 8310 genes were differentially expressed in the comparisons of CKvsM10, CKvsM50, CKvsM100, respectively. The analysis of differentially expressed genes (DEGs) showed phenylpropanoid biosynthesis, vitamin B6, ascorbate and aldarate metabolism-related genes were significantly enriched. The transcripts of flavonoid and terpenoid metabolism pathways and plant hormone signal transduction, especially the jasmonic acid (JA) signal-related genes, were differentially expressed in CKvsM50 and CKvsM100 comparisons. In addition, the transcription factors (TFs), e.g., MYC2 , DELLA , MYB111 played a key role in rosemary suspension cells under MeJA treatments. qRT-PCR of eleven DEGs showed a high correlation between the RNA-seq and the qRT-PCR result. Taken together, MeJA alleviated peroxidative damage of the rosemary suspension cells in a wide concentration range via concentration-dependent differential expression patterns. This study provided a transcriptome sequence resource responding to MeJA and a valuable resource for the genetic and genomic studies of the active compounds engineering in rosemary.

The effect of spontaneous fermentation by lactic acid bacteria on the extraction yield of bioactive compounds and antioxidant activity from rosemary leaf extracts was investigated using high-performance thin-layer chromatography (HPTLC). Brining and spontaneous fermentation with lactic acid bacteria more than doubled extraction of polyphenolics and antioxidants from the rosemary leaves. The results show that lactic acid fermentation enhances antioxidant activity in extracts by increasing the total phenolic content but does not increase extraction of phytosterols. Increased extraction of phenolic oxidants during fermentation assisted extraction, results from the in situ generated natural eutectic solvent from the plant sample. ATR-FTIR spectra from the bioactive bands suggests that this increased antioxidant activity is associated with increased extraction of rosmarinic acid, depolymerised lignin, abietane diterpenoids and 15-hydroxy-7-oxodehydroabietic acid.

Background: Chromium hexavalent (CrVI) is known as a toxic contaminant that induced oxidative stress and nephrotoxicity in humans and animals. Rosmarinus officinalis is a perennial herb rich in biologically active constituents that have powerful antioxidant properties. So, the current work evaluated the effectiveness of Rosmarinus officinalis essential oil (REO) against alterations induced by potassium dichromate in the kidney of male rats., Methods: GC-MS analysis, in vitro total phenol contents, and DPPH scavenging activity of REO were estimated. Thirty-five Wistar male rats were categorized into 5 groups. The first group was the control, the second one was orally administered rosemary essential oil (REO; 0.5 mL/kg BW), the third group was injected intraperitoneally with hexavalent chromium (CrVI; 2 mg/kg BW) for 14 days, the fourth group used as the protective group (REO was administrated 30 min before i.p. injection of CrVI) and the fifth group applied as the therapeutic group (rats injected with CrVI 30 min followed by oral administration of REO), respectively., Results: Twenty-nine components were detected with high total phenolic contents and high DPPH scavenging activity. Results revealed that CrVI- intoxicated rats showed a valuable increase in oxidative stress profile (TBARS and H 2 O 2 ) and a notable decline in glutathione (GSH), total protein content, and enzymatic antioxidants (SOD, CAT, GPx, and GST). Furthermore, serum kidney functions biomarkers (urea, creatinine, and uric acid) were increased significantly. Also, the administration of CrVI showed histological and immunohistochemical (PCNA-ir) changes in rat kidney tissue. Otherwise, administration of REO pre or post-treatment with CrVI significantly restored most of the biochemical parameters in addition to improvement in kidney tissue architecture. Moreover, individual intake with REO exhibited an amendment in oxidative stress markers., Conclusion: Conclusively, REO had a potential antioxidant capacity in ameliorating K 2 Cr 2 O 7 -induced nephrotoxicity, especially in the protection group., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Whether chemists or biologists, researchers dealing with metabolomics require tools to decipher complex mixtures. As a part of metabolomics and initially dedicated to identifying bioactive natural products, dereplication aims at reducing the usual time-consuming process of known compounds isolation. Mass spectrometry and nuclear magnetic resonance are the most commonly reported analytical tools during dereplication analysis. Though it has low sensitivity, 13 C NMR has many advantages for such a study. Notably, it is nonspecific allowing simultaneous high-resolution analysis of any organic compounds including stereoisomers. Since NMR spectrometers nowadays provide useful data sets in a reasonable time frame, we have embarked upon writing software dedicated to 13 C NMR dereplication. The present study describes the development of a freely distributed algorithm, namely MixONat and its ability to help researchers decipher complex mixtures. Based on Python 3.5, MixONat analyses a { 1 H}- 13 C NMR spectrum optionally combined with DEPT-135 and 90 data-to distinguish carbon types (i.e., CH 3 , CH 2 , CH, and C)-as well as a MW filtering. The software requires predicted or experimental carbon chemical shifts (δc) databases and displays results that can be refined based on user interactions. As a proof of concept, this 13 C NMR dereplication strategy was evaluated on mixtures of increasing complexity and exhibiting pharmaceutical (poppy alkaloids), nutritional (rosemary extracts) or cosmetics (mangosteen peel extract) applications. Associated results were compared with other methods commonly used for dereplication. MixONat gave coherent results that rapidly oriented the user toward the correct structural types of secondary metabolites, allowing the user to distinguish between structurally close natural products, including stereoisomers.

Biosynthesis of carnosic acid (CA), one of the most industrially valuable medicinal compounds present in rosemary (Rosmarinus officinalis L.) leaves, is affected by various plant stressors. In this study, effects of silver nanoparticle (AgNP) treatment on the secondary metabolism and CA production of rosemary plants were investigated. AgNP of 0, 25, 50, 100, and 200 ppm were utilized on hydroponically grown plants using foliar spray. Efficient absorbance and translocation of AgNPs to the plant roots were confirmed by XRF (X-ray fluorescence) analysis. The fluctuations of important antioxidant compounds such as CA content, phenolics, flavonoids, and acid ascorbic were analyzed and their correlations evaluated. Results revealed that application of 200 ppm AgNPs for 12 days increased CA level more than 11%, as compared to the control plants. Furthermore, significant positive correlations were observed between total flavonoids and CA content under AgNP treatment, suggesting that AgNP acted as an elicitor and triggered the enhancement of CA accumulation effectively. These data suggest that concentration-dependent AgNP may be used to boost antioxidant activity and phytochemical contents of other medicinal plants.

Element contents in different types of spices and aromatic herbs collected from Italy and Tunisia were evaluated. The aim was to evaluate: the possible differences and/or similarities among the analyzed samples; if it is possible related the samples to their geographical origins; the nutritional quality and the potential health risks. Potassium, Ca, Mg and Ni were low in laurel and rosemary; mint and thyme showed the highest Na and the lowest Se contents; arsenic and Cd levels were found highest in verbena which had also the lowest Hg content; lastly, black pepper had the highest Mn and the lowest Pb contents. Instead discrimination between Italian and Tunisian samples for each spices and aromatic herbs under analysis was achieved by PCA. Essential elements intake through samples was small. Levels of Pb in some samples exceed the maximum allowable level, but any safety risk for consumers is excluded., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Rosemary and sage species from Lamiaceae contain high amounts of structurally related but diverse abietane diterpenes. A number of substances from this compound family have potential pharmacological activities and are used in the food and cosmetic industry. This has raised interest in their biosynthesis. Investigations in Rosmarinus officinalis and some sage species have uncovered two main groups of cytochrome P450 oxygenases that are involved in the oxidation of the precursor abietatriene. CYP76AHs produce ferruginol and 11-hydroxyferruginol, while CYP76AKs catalyze oxidations at the C20 position. Using a modular Golden-Gate-compatible assembly system for yeast expression, these enzymes were systematically tested either alone or in combination. A total of 14 abietane diterpenes could be detected, 8 of which have not been reported thus far. We demonstrate here that yeast is a valid system for engineering and reconstituting the abietane diterpene network, allowing for the discovery of novel compounds with potential bioactivity.

Arsenic (As) recognized as a group I human carcinogen additionally poses a threat to plants which limit growth, metabolic activity, and productivity. Melatonin (MEL) is a naturally occurring compound in plants that have been recognized to mediate numerous morphological, physiological and molecular processes. Conversely, the role of MEL in inducing As-tolerance remains inexpressible and the plausible mechanisms in inducing As tolerance have remained largely unknown. The present investigation was designed to understand the protective role of MEL concentrations in rosemary herbs cultivated under As contamination. Arsenic evoked a deleterious decline on herb productivity, photosynthetic pigment, ion concentration, water status, ascorbic acid, essential oil (EO) yield and induced malformation of the chloroplast. Alternatively, increased organic osmolytes, oxidative impairment criteria, additionally antioxidant enzymes, phenol, flavonoid, anthocyanin, and EO%. Exogenous application of MEL with or without As, considerably increased growth, photosynthetic pigment, ion concentration, organic osmolytes as well as EO yield regarding polluted or non-polluted treatment respectively. Moreover, MEL treatment stabilized the cell membrane integrity, suppressed oxidative impairment criteria, and enhanced antioxidant capacity, additionally upregulation antioxidant enzymes. Plant treated with As showed a significant increase in As contamination and a bioconcentration factor in both root and shoot system. MEL supplementation under normal or As concentration, reduced As accumulation and bioconcentration factors, in either shoot or root systems. Additionally As decrease transfer factor, however, supplementation of MEL further decreased it. Application of 50 μM MEL might help the herbs to withstand As stress by strengthening their antioxidant machinery and osmoregulation capacity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.

The oxidative stability of Echium oil has been studied by two accelerated oxidation methodologies namely Rancimat and differential scanning calorimetry at selected temperatures in the range 50-110 °C. Kinetic analyses have provided Arrhenius parameters, oxidative stability indexes (OSI), temperature coefficients (t coef ), Q 10 and OSI at 20 °C (OSI 20 ) for both methodologies focussing on secondary and tertiary oxidation products. Peroxide, p-anisidine and polar compounds values have been measured to characterize from primary to tertiary oxidation products. In general, susceptibility of this oil to oxidation has been found to be larger than for other vegetable oils, according to the OSI 20 values estimated by Rancimat and differential scanning calorimetry. However, the values of t coeff and Q 10 are found to be similar to those described for vegetable oils. In addition, a study of the influence of two antioxidants, namely hydroxytyrosol and rosemary extract on the oxidative stability of Echium oil has been carried out using both methodologies. Protective effects over oxidation by up to a threefold increase of OSI 20 values have been found for both antioxidants at the highest dose utilized., (Copyright © 2019. Published by Elsevier Ltd.)

During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system's overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants' monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions' structure., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The study reports on the effect of anaerobic digestate derived composts on the metabolite composition and thermal behaviour of rosemary (Rosmarinus officinalis L.). Plants were cultivated in semiarid soil under four different fertiliser treatments (composts of anaerobic digested cattle (C) or pig slurry (P) at 30t/ha and 60 t/ha, and two control treatments (inorganic fertiliser and no fertiliser application). Samples of leaves and stems were analysed to investigate the effect of treatment on chemical composition and thermochemical properties. Three orthogonal analytical approaches were used, namely: Fourier transform mid infrared spectroscopy (FTIR), gas chromatography/mass spectrometry (GC/MS) and thermochemical gravimetric analysis (TGA). FTIR and GC/MS showed fertiliser treatment resulted in tissue specific changes in sample metabolite composition. Fertiliser treatment was detected to change the thermogravimetric properties of the leaf samples and from inorganic and composted pig slurry digestate treatments had greater ash content and lower proportions of fixed carbon compared with samples from the unfertilised control treatment. This study provides information on how the composition of rosemary might be altered by fertiliser application in regions of poor soil, and has implications for biomass quality when rosemary is grown on semi-wild sites for the purpose of soil improvement.

In this study, glazing with water and rosemary (Rosmarinus officinalis) extract were applied on frozen mud shrimp (Solenocera melantho) and stored at -20 °C for 24 weeks. Quality loss and protein and lipid changes of shrimp were evaluated by total volatile basis nitrogen (TVB-N), drip loss, moisture distribution, sulfhydryl content (SH), disulfide bond, intrinsic fluorescence intensity, lipid content, free fatty acids (FFA), peroxide value (PV), fluorescent compounds and sensory characteristics. Results showed that unglazed mud shrimp exhibited significant quality decline after 16 weeks of frozen storage. Glazing treatment significantly reduced quality loss, protein degradation, and lipid oxidative damage of shrimp during the 24 weeks of frozen storage, compared to the unglazed control sample. Glazing with rosemary extract was more effective in controlling quality changes in frozen mud shrimp with lower TVB-N, drip loss, PV, FFA and higher lipid content and sensory scores., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against oral infections. The aim of this study was to investigate the antimicrobial and antibiofilm activity of Mediterranean herb extracts against representative oral microorganisms. The extraction procedures and the analysis of the obtained extracts were performed under established experimental conditions. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of the methanol extracts of Cistus creticus ssp. creticus, Cistus monspeliensis, Origanum vulgare, Rosmarinus officinalis, Salvia sclarea and Thymus longicaulis against eight typical oral bacteria and the fungus Candida albicans were determined. The antibiofilm activity against Streptococcus mutans was also quantified using the microtiter plate test. Overall, all tested extracts inhibited effectively the screened obligate anaerobic microorganisms and in concentrations ≥0.3 mg ml-1 had moderate to high antibiofilm activity comparable to that of chlorhexidine (CHX) against S. mutans. In particular, R. officinalis (MIC: 0.08-5.00 mg ml-1) and S. sclarea (MIC: 0.08-2.50 mg ml-1) showed the highest antibacterial activity, while Cistus spp., R. officinalis and S. sclarea significantly inhibited S. mutans biofilm formation at 0.60, 1.25 and 2.50 mg ml-1, respectively. Porphyromonas gingivalis and Parvimonas micra were high susceptible to O. vulgare (MIC = 0.30 mg ml-1), whereas T. longicaulis eradicated all oral bacteria (MBC: 0.15-2.50 mg ml-1). Nevertheless, C. albicans showed no sensitivity to the tested extracts. In conclusion, the tested plant extracts could serve as alternative natural antibacterial and antibiofilm components against oral infections., Competing Interests: The authors have declared that no competing interests exist.

Background: Rosmarinus officinalis is an aromatic plant used in folk medicine as a result of the therapeutic properties associated with its phenolic composition, being rich in rosmarinic acid (RA) and caffeic acid (CA). To better understand the bioactivity of these compounds, their absorption and metabolism were assessed in human Caco-2 and HepG2 cells, as small intestine and liver models, respectively, using RA and CA standards, as well as a rosemary infusion and ferulic acid (FA)., Results: Test compounds were partially up-taken and metabolized by Caco-2 and HepG2 cells, although a higher metabolization rate was observed after hepatic incubation compared to intestinal incubation. CA was the compound best absorbed followed by RA and FA, showing metabolites percentages of 30.4%, 11.8% and 4.4% in Caco-2 and 34.3%, 10.3% and 3.2% in HepG2 cells, respectively. RA in the rosemary infusion showed improved bioavailability compared to pure RA. Methyl derivatives were the main metabolites detected for CA and RA after intestinal and hepatic metabolism, followed by methyl-glucuronidates and glucuronidates. RA was also minimally hydrolyzed into CA, whereas FA only was glucuronidated. Rosemary polyphenols followed the same biotransformation pathways as the standards. In addition, phase II derivatives of luteolin were observed., Conclusion: Rosemary polyphenols are partially metabolized in both the intestine and liver. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

There are more than 1300 articles in scientific literature dealing with positive impacts of Ultrasound-Assisted Extraction (UAE) such as reduction of extraction time, diminution of solvent and energy used, enhancement in yield and even selectivity, intensification of diffusion, and eliminating wastes. This present study aims to understand what are the mechanism(s) behind these positive impacts which will help to design a decision tool for UAE of natural products. Different microscopic observations (Scanning Electron Microscopy (SEM), Environmental Scanning Electron Microscopy (e-SEM), Cyto-histochemistry) have been used for spacial and temporal localization of metabolites in rosemary leaves, which is one of the most studied and most important plant for its antioxidant metabolites used in food industry, during conventional and ultrasound extraction. The study permits to highlight that ultrasound impacted rosemary leaves not by a single or different mechanisms in function of ultrasound power, as described by previous studies, but by a chain detexturation mechanism in a special order: local erosion, shear forces, sonoporation, fragmentation, capillary effect, and detexturation. These detexturation impacts followed a special order during ultrasound treatment leading at the end to the total detexturation of rosemary leaves. These mechanisms and detexturation impacts were identified in glandular trichomes, non-glandular-trichomes and the layer adaxial and abaxial cuticle. Modelling metabolites diffusion phenomenon during conventional and ultrasound extraction with the second Fick's law allowed the estimation of diffusivities and solvent penetration into the inner tissues and in meantime to accelerate the release of valuable metabolites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Our skin, our belongings, the world surrounding us, and the environment we live in are covered with molecular traces. Detecting and characterizing these molecular traces is necessary to understand the environmental impact on human health and disease, and to decipher complex molecular interactions between humans and other species, particularly microbiota. We recently introduced 3D molecular cartography for mapping small organic molecules (including metabolites, lipids, and environmental molecules) found on various surfaces, including the human body. Here, we provide a protocol and open-source software for 3D molecular cartography. The protocol includes step-by-step procedures for sample collection and processing, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, quality control (QC), molecular identification using MS/MS, data processing, and visualization with 3D models of the sampled environment. The LC-MS method was optimized for a broad range of small organic molecules. We enable scientists to reproduce our previously obtained results, and illustrate the broad utility of our approach with molecular maps of a rosemary plant and an ATM keypad after a PIN code was entered. To promote reproducibility, we introduce cartographical snapshots: files that describe a particular map and visualization settings, and that can be shared and loaded to reproduce the visualization. The protocol enables molecular cartography to be performed in any mass spectrometry laboratory and, in principle, for any spatially mapped data. We anticipate applications, in particular, in medicine, ecology, agriculture, biotechnology, and forensics. The protocol takes 78 h for a molecular map of 100 spots, excluding the reagent setup.

Rosemary extract is used in food additives and traditional medicine and has been observed to contain anti-tumor activity. In this study, rosemary extract is hypothesized to induce synthetic lethality in BRCA2 deficient cells by PARP inhibition. Chinese hamster lung V79 cells and its mutant cell lines, V-C8 (BRCA2 deficient) and V-C8 with BRCA2 gene correction were used. Rosemary extract and its major constituent chemicals were tested for their cytotoxicity by colony formation assay in cells of different BRCA2 status. The latter chemicals were tested for inhibitory effect of poly (ADP-ribose) polymerase (PARP) activity in vitro and in vivo. Rosemary has shown selective cytotoxicity against V-C8 cells (IC 50 17 µg/ml) compared to V79 cells (IC 50 26 µg/ml). Among tested chemicals, gallic acid and carnosic acid showed selective cytotoxicity to V-C8 cells along with PARP inhibitory effects. Carnosol showed comparative PARP inhibitory effects at 100 µM compared to carnosic acid and gallic acid, but the selective cytotoxicity was not observed. In conclusion, we predict that within rosemary extract two specific constituent components; gallic acid and carnosic acid were the cause for the synthetic lethality.

Carnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary ( Rosmarinus officinalis ). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis ( Arabidopsis thaliana ) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Phytogenic feed additives represent a potential alternative to antibiotics with attributed health and growth-promoting effects. Chickens supplemented with an essential oil blend, a Quillaja saponin blend, or a combination of both phytogenic preparations showed a comprehensively and significantly improved apparent ileal digestibility of crude protein and amino acids compared to control birds. Accordingly, holistic transcriptomic analyses of jejunum and liver samples indicated alterations of macromolecule transporters and processing pathways likely culminating in an increased uptake and metabolizing of carbohydrates and fatty acids. Complementary analyses in Caco-2 showed a significant increase in transporter recruitment to the membrane (SGLT1 and PEPT1) after addition of essential oils and saponins. Although the penetrance of effects differed for the used phytogenic feed additives, the results indicate for an overlapping mode of action including local effects at the intestinal border and systemic alterations of macronutrient metabolism resulting in an improved performance of broilers.

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease, characterized by inflammation of synovial joints. Carnosic acid (CA) is a phenolic diterpene isolated from Rosmarinus officinailis, playing a central role in cytoprotective responses to oxidative stress and inflammation response. Our study aimed to investigate the effects of CA on RA progression in diabetic animals. Carnosic acid (CA) was used to treat collagen-induced arthritis (CIA)-induced db/db mice. Blood glucose, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were investigated to explore insulin resistance. CA significantly down-regulated fasting blood glucose, glucose level in OGTT and ITT, ameliorated CIA-induced bone loss, and reduced pro-inflammatory cytokines and reactive oxygen species (ROS) in db/db mice with arthritis induced by CIA. In vitro, CA suppressed Receptor Activator for Nuclear Factor-κ B Ligand (RANKL)- and Macrophage colony-stimulating factor (M-CSF)-induced osteoclastogenesis. The osteoclastic specific markers were inhibited by CA. Signal transduction studies showed that CA significantly decreased the expression of molecules contributing to ROS and increased anti-oxidants. Additionally, CA inactivated the RANKL- and M-CSF-induced p38 mitogen activated protein kinases (MAPK), inhibited NF-κB phosphorylation, causing pro-inflammatory cytokines down-regulation. Together, CA ameliorated osteoclast formation and CIA-induced bone loss in db/db mice through inflammation suppression by regulating ROS-dependent p38 pathway., (Copyright © 2017. Published by Elsevier Inc.)

Solar irradiation induces skin inflammatory processes causing deleterious effects like premature ageing. In this study, the designed lipoid colloidal carrier (LCC) was loaded with Diosmin in combination with different essential oils, to be used as a topical photo-protective preparation. To investigate the ability of the essential oils to potentiate Diosmin effects, the Diosmin/essential oil-loaded LCCs (LCC2, LCC3 and LCC4) were compared to the Diosmin-loaded LCC (LCC1). The incorporated essential oils were those of Rosmarinus officinalis, Zingiber officinale or Vitis vinifera in LCC2, LCC3 and LCC4, respectively. All the LCCs had particle size (PS) values ranging from 121.1 to 144.3nm with uniform distribution and, zeta potential (Z) values around 30mV. Also, they all had high drug encapsulation efficiencies. LCC1 had the lowest anti-oxidant and in-vitro sun-blocking effect (p<0.05). In-vivo photo-protective studies showed that all the formulated LCCs had a skin protective effect when compared to the positive control (p<0.05); however LCC1 had the lowest anti-erythemal and anti-wrinkling effect. Histological studies proved the efficacy of the designed LCCs as skin anti-photoageing, with LCC1 showing the lowest anti-inflammatory and anti-wrinkling effect, while LCC2 had the highest anti-wrinkling effect. These results indicated that the suggested Diosmin/essential oil combinations improved the anti-oxidant, sun-blocking and anti-photoageing effects of Diosmin. After one year of storage, the LCCs showed satisfactory physical stability. This study presents the designed LCCs as safe and effective nano-structured dermal care products containing 'all-natural' components., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to improve the oxidative stability of soybean oil by using aromatic plants. Soybean oil flavored with rosemary (ROS) and soybean oil flavored with thyme (THY) were subjected to heating for 24h at 180°C. The samples were analyzed every 6h for their total polar compounds, anisidine values, oxidative stability and polyphenols content. The tocopherols content was determined and volatile compounds were also analyzed. After 24h of heating, the incorporation of these plants using a maceration process reduced the polar compounds by 69% and 71% respectively, in ROS and THY compared to the control. Until 6h of heating, the ROS kept the greatest oxidative stability. The use of the two extracts preserves approximately 50% of the total tocopherols content until 18h for the rosemary and 24h for the thyme flavored oils. Volatile compounds known for their antioxidant activity were also detected in the formulated oils. Aromatic plants added to the soybean oil improved the overall acceptability of potato crisps (p<0.05) until the fifteenth frying., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial "chassis" have provided access to important industrial compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis.Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies.

Coal tar is a significant product generated from coal pyrolysis. Coal tar can be utilized as raw materials for various industries. It is also a type of raw material from which phenols, naphthalenes, and anthracene can be extracted. The present study was designed to investigate the possibility of coal tar creosote to induce oxidative stress and biochemical perturbations in rat liver and the role of rosemary (Rosmarinus officinalis) in ameliorating its toxic effects. Male Wister Albino rats were randomly divided into four groups of seven each, group I served as control; group II treated with rosemary (10 mL of water extract/kg BW for 21 days), group III received coal tar creosote (200 mg/4 mL olive oil/kg BW for 3 days), and group IV treated with both rosemary and coal tar creosote. The administration of coal tar creosote significantly caused elevation in lipid peroxidation (LPO) and reduction in the activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). A significant decrease in reduced glutathione (GSH) content was also observed. Liver aminotransferases aspartate transaminase (AST) and alanine transaminase (ALT)] and alkaline phosphatase (AlP) were significantly decreased while lactate dehydrogenase (LDH) was increased. Rosemary pretreatment to coal tar creosote-treated rats decreased LPO level and normalized GPx, GR, SOD, CAT, and GST activities, while GSH content was increased. Also, liver AST, ALT, AlP, and LDH were maintained near normal level due to rosemary treatment. In conclusion, rosemary has beneficial effects and could be able to antagonize coal tar creosote toxicity., (© 2014 Wiley Periodicals, Inc.)

Enterotoxigenic Staphylococcus aureus strains that were isolated from foods were investigated for their ability to develop direct-tolerance and cross-tolerance to sodium chloride (NaCl), potassium chloride (KCl), lactic acid (LA) and acetic acid (AA) after habituation in sublethal amounts (1/2 of the minimum inhibitory concentration - 1/2 MIC and 1/4 of the minimum inhibitory concentration - 1/4 MIC) of Origanum vulgare L. essential oil (OVEO). The habituation of S. aureus to 1/2 MIC and 1/4 MIC of OVEO did not induce direct-tolerance or cross-tolerance in the tested strains, as assessed by modulation of MIC values. Otherwise, exposing the strains to OVEO at sublethal concentrations maintained or increased the sensitivity of the cells to the tested stressing agents because the MIC values of OVEO, NaCl, KCl, LA and AA against the cells that were previously habituated to OVEO remained the same or decreased when compared with non-habituated cells. These data indicate that OVEO does not have an inductive effect on the acquisition of direct-tolerance or cross-tolerance in the tested enterotoxigenic strains of S. aureus to antimicrobial agents that are typically used in food preservation.

Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a naturally occurring hydroxylated compound commonly found in species of the subfamily Nepetoideae of the Lamiaceae and Boraginaceae, such as Rosmarinus officinalis, Salvia officinalis, and Perilla frutescens. RA is biosynthesized from the amino acids L-phenylalanine and L-tyrosine by eight enzymes that include phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. RA can also be chemically produced by the esterification of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA and its numerous derivatives containing one or two RA with other aromatic moieties are well known and include lithospermic acid, yunnaneic acid, salvianolic acid, and melitric acid. Recently, RA and its derivatives have attracted interest for their biological activities, which include anti-inflammatory, anti-oxidant, anti-angiogenic, anti-tumor, and anti-microbial functions. Clinically, RA attenuates T cell receptor-mediated signaling, attenuates allergic diseases like allergic rhinitis and asthma, and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like symptoms, protects from neurotoxicity, and slows the development of Alzheimer's disease. These attributes have increased the demand for the biotechnological production and application of RA and its derivatives. The present review discusses the function and application of RA and its derivatives including the molecular mechanisms underlying clinical efficacy.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Honey contains a variety of polyphenols and represents a good source of antioxidants, while the human diet often contains compounds that can cause DNA damage. The present study investigated the protective effect of three commercial honey samples of different floral origin (rosemary, heather and heterofloral) from Madrid Autonomic Community (Spain) as well as an artificial honey on DNA damage induced by dietary mutagens, using a human hepatoma cell line (HepG2) as in vitro model system and evaluation by the alkaline single-cell gel electrophoresis or comet assay., Results: Rosemary, heather and heterofloral honeys protected against DNA strand breaks induced by N-nitrosopyrrolidine (NPYR), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but none of the honey samples tested prevented DNA strand breaks induced by N-nitrosodimethylamine (NDMA). Heterofloral and heather (unifloral) honeys with higher phenolic content were most effective in protecting HepG2 cells against DNA damage induced by food mutagens. Heterofloral honey was more protective against NPYR and BaP, while heather honey was more protective against PhIP. Artificial honey did not show a protective effect against DNA damage induced by any of the food mutagens tested, indicating that the protective effects of honeys could not be due to their sugar components., Conclusion: The results suggest that the protective effect of three kinds of Spanish honey of different floral origin could be attributed in part to the phenolics present in the samples. Honeys with higher phenolic content, i.e. heather and heterofloral honeys, were most effective in protecting against food mutagen-induced DNA damage in HepG2 cells. In addition, a possible synergistic effect between other minor honey components could also be involved., (© 2013 Society of Chemical Industry.)

The aim was to assess the in vitro effect of pasteurized egg (PE) and rosemary (Rosmarinus officinalis) on frozen-thawed ram semen. Ejaculates from three mature rams of the Rasa Aragonesa breed were cryopreserved using a 2-step dilution method (Fraction 1: F1; Fraction 2: F2). In Experiment 1, semen was frozen in egg yolk (EY) or PE extenders. After thawing, similar results were obtained in terms of total and progressive motility, viability, hypo-osmotic swelling test (HOST) and acrosome integrity after 2 h incubation. In Experiment 2, addition of rosemary to F1, F2 or both fractions to EY extenders was evaluated. Rosemary in F1 decreased progressive motility (p = 0.013) after 2 h incubation. Finally, PE can be used as a substitute for EY to reduce hygienic risks in extenders and is easier to standardize. Supplementation of EY extender with rosemary in F1 reduced progressive motility. Rosemary supplementation in F2 does not affect semen quality.

This study aimed at estimating exposition risks to wild rosemary used as herbs in the contaminated area of the former smelting factory of L'Escalette (South of Marseille, France). Metals and metalloids i.e. Pb, As, Sb, Zn, and Cu concentrations were analyzed in soils and in rosemary aerial parts (stems and leaves) on two sites: one heavily contaminated and the other far away from the pollution source, considered as reference. The metal and metalloid transfer into water during the brewing process of herbal tea was also determined. A mixed contamination by the above-cited contaminants was demonstrated in soils of the factory site, with average concentrations of 9253, 1127, 309, 2698 and 32 mg/kg for Pb, As, Sb, Zn and Cu, respectively. However, metals and metalloids' transfer in rosemary aerial parts was limited, as bioaccumulation factors were under 1. Thus, Pb, As and Cu concentrations in leaves were below international regulation limits concerning ingestion of medicinal herbs (no regulation values available for Sb and Zn). This study highlighted that, if contaminated rosemary leaves were ingested, health risks may be limited since acceptable daily intake (ADI) for Pb, As, Sb and Cu (no ADI value available for Zn) will only be reached if very high quantities are consumed. Furthermore, we aimed to establish if this mixed contamination could alter rosemary's essential oil quality, and thereby the compositions of essential oils obtained from individuals on the heavily contaminated soil were compared to those obtained from the reference population. An increased biosynthesis of antioxidant compounds was favored in essential oils from rosemary individuals growing in contaminated site. Although the health risk of a long-term exposition of low level of the mixed contamination by rosemary ingestion is not easy to elucidate, the use of rosemary essential oils from contaminated site appears as safe., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To assess metal mobility in pruning waste and biosolids compost (pH 6.9 and total concentration of metals in milligram per kilogram of Cd 1.9, Cu 132, Fe 8,513, Mn 192, Pb 81, and Zn 313), shrubs species Atriplex halimus and Rosmarinus officinalis were transplanted in this substrate and irrigated with citric acid (4 g L(-1), pH 2.9) and nutrient solution daily for 60 days. Citric acid significantly increased the concentrations of soluble Mn and Fe in the nutrient substrate solution measured by suction probes, while other metals did not vary in concentration (Cu and Zn) or were not observed at detectable levels (Cd and Pb). In plants, citric acid significantly increased the concentrations of Cu (2.7 ± 0.1-3.3 ± 0.1 mg kg(-1)), Fe (49.2 ± 5.2-76.8 ± 6.8 mg kg(-1)), and Mn (7.2 ± 1.1-11.4 ± 0.7 mg kg(-1)) in leaves of R. officinalis, whereas the concentration of only Mn (25.4 ± 0.3-42.2 ± 2.9 mg kg(-1)) was increased in A. halimus. Increasing Fe and Mn solubility by citric acid addition indicates the possibility of using it to improve plant nutrition. The mobility of metals in this substrate was influenced for the concentration of the metal, the degree of humification of organic matter and its high Fe content.

This study aimed at identifying the extent and type of contamination of a former lead smelting site in the area of Marseille, France, dating from the industrial revolution, and to evaluate environmental hazards and opportunities for phytoremediation, a promising sustainable technology. Amongst the native plants growing in this semiarid shrub ecosystem, two perennials Globularia alypum L. and Rosmarinus officinalis L. were selected. Twenty-one soil/plant couples were collected and seventeen additional soil samples were added to better characterize the soil pollution of the area. A multi-contamination by Pb, As, Sb, Zn, Cu was demonstrated, with huge variations within the contamination levels. The soils highest concentrations were encountered along the horizontal chimney and on the slag heaps area. However, both sites differed from each other. The former was characterized by the highest Pb, As and Sb concentrations that could reach 130, 7.0 and 9.0gkg(-1) respectively, the latter, by high Cu, Fe, Mn, S concentrations, even if it was also heavily contaminated by Pb and Zn. G. alypum and R. officinalis were shown to be metal-tolerant and to accumulate trace metals and As. Due to the low bioconcentration and translocation factors determined, both species may not be used for phytoextraction, but seem to be good candidates for phytostabilization., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ionic liquid based, ultrasound-assisted extraction was successfully applied to the extraction of phenolcarboxylic acids, carnosic acid and rosmarinic acid, from Rosmarinus officinalis. Eight ionic liquids, with different cations and anions, were investigated in this work and [C(8)mim]Br was selected as the optimal solvent. Ultrasound extraction parameters, including soaking time, solid-liquid ratio, ultrasound power and time, and the number of extraction cycles, were discussed by single factor experiments and the main influence factors were optimized by response surface methodology. The proposed approach was demonstrated as having higher efficiency, shorter extraction time and as a new alternative for the extraction of carnosic acid and rosmarinic acid from R. officinalis compared with traditional reference extraction methods. Ionic liquids are considered to be green solvents, in the ultrasound-assisted extraction of key chemicals from medicinal plants, and show great potential.

1. The current study was conducted to evaluate the effects of dietary supplementation with vitamin E (as alpha-tocopherol acetate), dried rosemary leaves and rosemary volatile oil on the performance, meat quality (measured as sensory variables, pH, colour, malondialdehyde (MDA) level, and bacteria count) and serum superoxide dismutase (SOD) activity in broilers fed on maize-soybean meal based diets. 2. A total of 800 broiler chicks were randomly allocated to 8 dietary treatments, which were set up with 1 control group and 7 experimental groups. The control group (VitE1) was given a basal diet including 50 mg/kg alpha-tocopherol acetate, while the experimental groups were given 5 x 7 g/kg rosemary plant (R1), 8 x 6 g/kg plant (R2), 11 x 5 g/kg plant (R3), 100 mg/kg plant oil (RO1), 150 mg/kg plant oil (RO2), 200 mg/kg plant oil (RO3) or 200 mg/kg alpha-tocopherol acetate (VitE2). 3. Although there were no statistical differences observed for feed consumption, other performance variables including live weight gain, feed efficiency and carcase yield were significantly affected. The addition of rosemary volatile oil had more effect on the performance variables than did the rosemary plant itself. 4. As a measure of meat shelf life, TBA analyses were performed on the meat samples on d 1, 3 and 5 after culling. Meat MDA levels of groups fed diets with rosemary and rosemary volatile oil were significantly lower than that of groups fed diets containing alpha-tocopherol acetate alone. 5. Significant differences were also seen between the control and experimental groups for meat colour and meat pH values as well as for sensory analyses. 6. Microbiological analyses conducted at the end of the experiment showed that E. coli counts were significantly reduced in meat samples from the experimental groups. 7. In conclusion, dietary supplementation with rosemary and its volatile oil improved broiler meat quality. Moreover growth performance was positively affected by the rosemary volatile oil supplementations.

The antioxidant constituents of essential oils (EOs) of Rosmarinus officinalis L. (α-pinene chemotype) were isolated at the flowering (A), post-flowering (B), and vegetative stages (C). GC-MS was used to analyze total chemical composition, Folin-Ciocalteau and Prussian blue methods for reducing substances. Radical scavenging capacity (DPPH test, IC(50) 36.78±0.38, 79.69±1.54, 111.94±2.56μL) and anti-lipoperoxidant activity (TBARS, IC(50) 0.42±0.04, 1.20±0.06μL, 4.07±0.05μL) differed widely in the three stages. Antioxidant activity, identified after silica gel fractionation chromatography, was closely related (R(2)=0.9959) to each EO's content of hydroxilated derivatives, (containing alcohols, phenols and 1,8 cineole): 15.26±0.12%, 7.22±0.06%, and 5.09±0.10% in EOs from A, B, and C. Modeling the C, H, O terpenes in a simulated phospholipid bilayer indicated that anti-lipoperoxidant activity depended on the stability and rapidity of their interactions with the membrane bilayer components, and their positioning over its surface., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The possibility of remediating contaminated soils though the use of high biomass-generating, native plant species capable of removing heavy metals is receiving increased attention. The cadmium (Cd) accumulation capacities of the native Mediterranean, perennial shrubs Atriplex halimus, Phyllirea angustifolia, Rhamnus alaternus and Rosmarinus officinalis were tested by growing transplanted specimens in a pine bark compost substrate (pH 5.6) contaminated with 100 mg Cd kg(-1). After 70 days, only R. alaternus showed reduced growth. The increase in biomass seen in all the test species enhanced the phytoextraction of Cd. However, the species behaved as metal excluders, except for the halophyte A. halimus, which behaved as an indicator plant. In this species the leaf Cd concentration reached 35 mg Cd kg(-1), with the shoot responsible for some 86% of total Cd accumulation. Atriplex halimus showed the highest bioconcentration factor (BCF) (0.36) and leaf Cd transport index (1.68); consequently, this species showed the highest Cd phytoextraction capacity.