New Insight into Justicidin B Pathway and Production in Linum austriacum.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

NMR Lipid Profile of Milk from Alpine Goats with Supplemented Hempseed and Linseed Diets.

The supplementation of goat diets with natural products to obtain milk with nutraceutical components is a common practice. In these last years, the influence of supplementation of specifically designed diets has been studied with different analytical tools in order to explore possible beneficial effects in human consumption of animal milk and milk-derived products. In this study, the lipid fraction of milk from Alpine goats undergoing different dietary regimens was studied by 1H-NMR spectroscopy. Alpine goats were fed with linseed or hempseed supplements, and after 14 weeks of treatment, milk was collected and analyzed. Results showed that feeding diets supplemented with seeds positively affected the fatty acid composition with a pronounced increase in unsaturated fatty acids for both diets compared to a control diet. Specifically, linolenic acid content was more than doubled for linseed diet compared with the hempseed and control groups, while linoleic acid greatly increased only upon hempseed supplementation. However, a number of conjugated linoleic acid (CLA) isomers and higher levels of fatty acids with trans configuration were found in supplemented diets, particularly in the linseed diet.

Cell Suspensions of Cannabis sativa (var. Futura): Effect of Elicitation on Metabolite Content and Antioxidant Activity.

Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp (C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts under the effect of specific elicitors.

Integrated analytical methodology to investigate bioactive compounds in Crocus sativus L. flowers.

INTRODUCTION: The increasing interest on Crocus sativus L. over the last decades is caused by its potential employment as a source of biologically active molecules, endowed with antioxidant and nutraceutical properties. These molecules are present mainly in stigmas and tepals, these last generally considered as byproducts. OBJECTIVE: To characterise bioactive compounds in stigmas, stamens, and tepals of Crocus sativus L. for quality, cross-contamination of tissues or fraudulent addition, joining spectroscopic and chromatographic techniques. METHODOLOGY: Fourier transform infrared (FT-IR) and Raman spectroscopies were initially employed, being very rapid in response; volatiles were more appropriately investigated by gas chromatography with mass spectrometry (GC-MS), while finally nuclear magnetic resonance (NMR) and high-performance liquid chromatography with a diode array detector (HPLC-DAD) were adopted for a more thorough characterisation of secondary metabolites. NMR was also used to investigate the anthocyanins content in tepals upon acid extraction. RESULTS: The results obtained highlighted the drying method as the dominant factor affecting the content of volatile constituents and contributing to the quality of saffron, while only slight differences were observed in the most abundant metabolites of stigmas, as well as in the anthocyanin content of tepals. In particular, for the first time, delphinidin and petunidin were detected by NMR in this latter tissue. CONCLUSION: The integrated analytical methodology here proposed, allowed to achieve a deeper level of compositional and structural details of secondary metabolites in Crocus sativus L. flowers.

Ultra-high performance liquid chromatography tandem mass spectrometry for the detection of durum wheat contamination or adulteration.

In this work, an ultra-performance liquid chromatography electrospray ionization (UPLC-ESI)-MS/MS methodology based on multiple reaction monitoring (MRM) for the selective and sensitive detection and quantification of durum wheat adulteration has been developed and fully validated. The targeted analysis was performed by monitoring specific transitions at m/z 543.7 > 657.4 and m/z 543.7 > 299.2 of a species-specific marker derived from a tryptic peptide of puroindoline a (Pin-a), a cysteine-rich protein selectively present only in common wheat. In addition, two transitions at m/z 500.4 > 725.4 and m/z 500.4 > 561.9 of a reference peptide belonging to purothionin A-1, present in both species, were also monitored. The calibration curves obtained on binary mixtures with known percentages of common/durum wheat flours showed linearity (coefficient of regression, r ≥ 0.99) over concentrations that ranged between 80 and 1%. The limit of detection (LOD) and limit of quantification (LOQ) for the Pin-a marker in wheat flours were 0.01 and 0.03%, respectively. The identified Pin-a marker was also found to be highly diagnostic for the quantification of common wheat in raw materials (kernels) and processed products (pasta), thus offering new opportunities to assess food authenticity.