Rosmarinus officinalis is a potent antioxidant herb rich in polyphenols. Ultra-high-performance liquid chromatography, coupled with electrospray ionization and quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS), enables an exhaustive, full-spectrum analysis of the molecular constituents of natural products. The study aimed to develop a rapid UHPLC method to contribute new insights into the phytochemical composition of rosemary and to assess the performance of nine different procedures for extraction. These include fresh tissue homogenization, fresh and dry leaf decoction, and their respective fermentation, Soxhlet extraction, and sonication using water and methanol. Different extraction methods were found to recover quite different groups of polyphenols within 11 min during 20 min of analysis. Soxhlet extraction, yielded very high concentrations of rosmarinic acid (33,491.33 ± 86.29 µg/g), luteolin-7-O-glucoside (209.95 ± 8.78 µg/g), carnosic acid (2915.40 ± 33.23 µg/g), carnosol (22,000.67 ± 77.39 µg/g), and ursolic acid (5144.27 ± 28.68 µg/g). UHPLC-ESI-QTOF-MS enabled the detection of more than 50 polyphenols, including phenolic acids, flavonoids, and terpenoids in the various extracts. Of these, sagerinic acid ([M − H]⁻m/z 719.16), salvianolic acid A ([M − H]⁻m/z 493.11) and B ([M − H]⁻m/z 717.15), and a pentacyclic triterpenoid corosolic acid ([M − H]⁻m/z 471.34) were detected for the first time in rosemary. Soxhlet extraction was found to be the most efficient method, followed by dry leaf decoction. The UHPLC-ESI-QTOF-MS methodology for the analysis proved to be very efficient in the identification and characterization of targeted and untargeted bioactive molecules in the rosemary.

UHPLC with QTOF-MS is widely used as a powerful tool for metabolomic analysis. This technology has recently been applied to the analysis of polyphenols in food and herb extracts. Sage (Salvia officinalis) and rosemary (Rosmarinus officinalis), belonging to the family Lamiaceae, are known for their potent antioxidant properties due to the presence of polyphenols. We have developed a sensitive and reproducible UHPLC-QTOF-MS/MS-based method for comprehensive phytochemical profiling and the identification and quantitation of specific polyphenolic compounds present in sage and rosemary leaves. The herbs were extracted ultrasonically using methanol as the solvent. In sage, rosmarinic acid (17 678.7±673.4 µg/g) and 12-methoxy carnosic acid (21 918.3±715.4 µg/g) were found in the highest concentrations among all polyphenols. In contrast, rosmarinic acid (14 311.0±636.4 µg/g), luteolin-3'-acetyl-O-glucuronide (1488.50±47.58 µg/g), and luteolin-7-O-glucuronide (1053.68±68.83 µg/g) were observed in the highest concentrations in rosemary. Sagerinic acid, rosmanol, rosmadial, carnosol, and carnosic acid were found in abundance in both sage and rosemary. The pentacyclic triterpenoid, corosolic acid ([M - H]¯ m/z 471.35), was detected for the first time in both plants. Of the 47 polyphenolic compounds identified in each plant, 38 compounds were found in common in rosemary and sage. A flavonoid compound, baicalin ([M -H]¯ m/z 445.08), was identified for the first time in S. officinalis. Also, pectolinarigenin ([M - H]¯ m/z 313.07), a dimethoxyflavone, was detected for the first time in both sage and rosemary leaves.

Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.

Background and Objectives: Stress can overload adaptive mechanisms, leading to epigenetic effects harmful to health. Research on the reversal of these effects is in its infancy. Early results suggest some meditation techniques have health benefits that grow with repeated practice. This study focused on possible transcriptomic effects of 38 years of twice-daily Transcendental Meditation^® (TM^®) practice. Materials and Methods: First, using Illumina^® BeadChip microarray technology, differences in global gene expression in peripheral blood mononuclear cells (PBMCs) were sought between healthy practitioners and tightly matched controls (n = 12, age 65). Second, these microarray results were verified on a subset of genes using quantitative polymerase chain reaction (qPCR) and were validated using qPCR in larger TM and control groups (n = 45, age 63). Bioinformatics investigation employed Ingenuity^® Pathway Analysis (IPA^®), DAVID, Genomatix, and R packages. Results: The 200 genes and loci found to meet strict criteria for differential expression in the microarray experiment showed contrasting patterns of expression that distinguished the two groups. Differential expression relating to immune function and energy efficiency were most apparent. In the TM group, relative to the control, all 49 genes associated with inflammation were downregulated, while genes associated with antiviral and antibody components of the defense response were upregulated. The largest expression differences were shown by six genes related to erythrocyte function that appeared to reflect a condition of lower energy efficiency in the control group. Results supporting these gene expression differences were obtained with qPCR-measured expression both in the well-matched microarray groups and in the larger, less well-matched groups. Conclusions: These findings are consistent with predictions based on results from earlier randomized trials of meditation and may provide evidence for stress-related molecular mechanisms underlying reductions in anxiety, post-traumatic stress disorder (PTSD), cardiovascular disease (CVD), and other chronic disorders and diseases.

Background: Our previous comparison of peripheral blood mononuclear cells (PBMCs) from long-term Transcendental Meditation^® (TM^®) practitioners and matched non-practitioner controls found 200 differentially expressed (DE) genes. Bioinformatics analyses of these DE genes suggested a reduced risk of diseases associated with stress and aging in the TM group. Here we assessed additional signs of reduced stress and aging. Methods: A sample of 15 of the 200 DE genes was studied using qPCR in PBMCs from 40-year TM practitioners (“Old TM”, n = 23) compared to a “Young Control” group (n = 19) and an “Old Control” group (n = 21) of non-meditators. In these three groups, plus a “Young TM”, 12-year practitioner group (n = 26), we also studied EEG-based parameters of cognitive function (the Brain Integration Scale (BIS), and latency of three components of the event-related potential (ERP)). Finally, using LC/MS/MS, we compared persistent levels of cortisol (F) and its inactive congener, cortisone (E), in hair. Results: qPCR analysis showed that 13 of the 15 genes were more highly expressed in Old Controls than in Young Controls. In the Old TM group, 7 of these 13 were lower than in Old Controls. Both TM groups had higher BIS scores than their age-matched controls. The Old TM group had shorter N2, P3a, and P3b latencies than the Old Control group, and latencies in the Old TM group were not longer than in the Young Control group. The Hair F/Hair E ratio was higher in the control subgroups than in their age-matched TM subgroups, and Hair F was higher in the Young Control and combined control groups than in the Young TM and combined TM groups. Conclusions: These results are consistent with reductions in biomarkers of chronic stress and biological age in long-term TM meditators. They are also consistent with results from the previous study suggesting that TM practice lowers energy consumption or leads to more efficient energy metabolism.

Synbio milk, containing recombinant bovine β-lactoglobulin (β-LG), produced in the fungus Trichoderma reesei, was deficient nutritionally compared to bovine milk. It also contained both fungal proteins and fungal metabolites never verified safe for human consumption. By three independent methods synbio milk was found to contain predominantly fungal protein, not the 90–99% β-LG, claimed by the product developer. By (1) shotgun proteomics, (2) ELISA and classical protein analysis, and (3) simulated mixing experiments, measuring percent deviation of the amino acid composition of synbio milk from that of bovine milk, fungal protein content was found to be 75.1%, 86.5% and 90–95%, respectively. Additionally, high sensitivity untargeted mass spectrometry revealed substantial levels of 69 nutrients in bovine milk, of which only 7 were present in small amounts in synbio milk. This analysis also revealed 93 compounds in synbio milk, byproducts/waste products of fungal fermentation, whose chemical identities could not be established searching large mass spectral databases, suggesting they are novel compounds. Neither these nor the fungal proteins found in the synbio milk have been tested for safety or allergenicity at exposure levels relevant for synbio milk consumption. Therefore, comprehensive toxicity and allergenicity testing are needed to assess the safety of synbio milk for human consumption.