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Lichen specific metabolites (LSMs) have interesting biological activities and quantitative variations may be present intraspecifically. For example, variations in medullary fumarprotocetraric acid (FA) and cortical usnic acid (UA) were observed in the lichen Cladonia foliacea, but the mechanism of variation is not well understood. The current study aimed to characterise the quantitative variation of FA and UA and to investigate the association between lichen metabolite content and ecological / biological variables. Fungal and algal trees were constructed using fungal (nrITS, RPB2) and algal (nrITS) loci, respectively. Using a chiral chromatographic method, the contents of (-)-UA were determined in 29 C. foliacea specimens and range from 6.88 to 34.27 mg/g dry wt. The FA contents were lower and varied from 1.44 to 9.87 mg/g dry wt. Although the fungal tree showed two well resolved clades, no significant differences of UA or FA contents were found between the two fungal clades. However, a significantly higher UA/FA ratio as well as a unique habitat were found to be associated with specimens which contained the alga Asterochloris lobophora than those specimens associated other Asterochloris algae. Taking all predictive variables into account (i.e. substrate type, elevation, collection season, photobiont identity), the multivariate data analysis indicated that photobiont identity explained most of the variance of LSM contents in C. foliacea. Thus future LSM biosynthetic studies should take the photobiont into consideration when dealing with intraspecific quantitative variation.

Astaxanthin derived from Haematococcus pluvialis is a valuable metabolite applied in a wide range of products. Its extraction depends on a sophisticated series of downstream process steps, including harvesting, disruption, drying, and extraction, of which some are dependent on each other. To determine the processes that yield maximum astaxanthin recovery, bead milling, high-pressure homogenization, and no disruption of H. pluvialis biomass were coupled with spray-drying, vacuum-drying, and freeze-drying in all possible combinations. Eventually, astaxanthin was extracted using supercritical CO2. Optimal conditions for spray-drying were evaluated through the design of experiments and standard least squares regression (feed rate: 5.8 mL/min, spray gas flow: 400 NL/h, inlet temperature: 180 °C). Maximal astaxanthin recoveries were yielded using high-pressure homogenization and lyophilization (85.4%). All combinations of milling or high-pressure homogenization and lyophilization or spray-drying resulted in similar recoveries. Bead milling and spray-drying repeated with a larger spray-dryer resulted in similar astaxanthin recoveries compared with the laboratory scale. Smaller astaxanthin recoveries after the extraction of vacuum-dried biomass were mainly attributed to textural changes. Evaluation of these results in an economic context led to a recommendation for bead milling and spray-drying prior to supercritical CO2 extraction to achieve the maximum astaxanthin recoveries.

IntroductionOats (Avena sativa L.) are a whole grain cereal recognised for their health benefits and which are cultivated largely in temperate regions providing both a source of food for humans and animals, as well as being used in cosmetics and as a potential treatment for a number of diseases. Oats are known as being a cereal source high in dietary fibre (e.g. β-glucans), as well as being high in antioxidants, minerals and vitamins. Recently, oats have been gaining increased global attention due to their large number of beneficial health effects. Consumption of oats has been proven to lower blood LDL cholesterol levels and blood pressure, thus reducing the risk of heart disease, as well as reducing blood-sugar and insulin levels.ObjectivesOats are seen as a low input cereal. Current agricultural guidelines on nitrogen application are believed to be suboptimal and only consider the effect of nitrogen on grain yield. It is important to understand the role of both variety and of crop management in determining nutritional quality of oats. In this study the response of yield, grain quality and grain metabolites to increasing nitrogen application to levels greater than current guidelines were investigated.MethodsFour winter oat varieties (Mascani, Tardis, Balado and Gerald) were grown in a replicated nitrogen response trial consisting of a no added nitrogen control and four added nitrogen treatments between 50 and 200 kg N ha−1 in a randomised split-plot design. Grain yield, milling quality traits, β-glucan, total protein and oil content were assessed. The de-hulled oats (groats) were also subjected to a rapid Ultra High Performance Liquid Chromatography—Mass Spectrometry (UHPLC-MS) metabolomic screening approach.ResultsApplication of nitrogen had a significant effect on grain yield but there was no significant difference between the response of the four varieties. Grain quality traits however displayed significant differences both between varieties and nitrogen application level. β-glucan content significantly increased with nitrogen application. The UHPLC-MS approach has provided a rapid, sub 15 min per sample, metabolite profiling method that is repeatable and appropriate for the screening of large numbers of cereal samples. The method captured a wide range of compounds, inclusive of primary metabolites such as the amino acids, organic acids, vitamins and lipids, as well as a number of key secondary metabolites, including the avenanthramides, caffeic acid, and sinapic acid and its derivatives and was able to identify distinct metabolic phenotypes for the varieties studied. Amino acid metabolism was massively upregulated by nitrogen supplementation as were total protein levels, whilst the levels of organic acids were decreased, likely due to them acting as a carbon skeleton source. Several TCA cycle intermediates were also impacted, potentially indicating increased TCA cycle turn over, thus providing the plant with a source of energy and reductant power to aid elevated nitrogen assimilation. Elevated nitrogen availability was also directed towards the increased production of nitrogen containing phospholipids. A number of both positive and negative impacts on the metabolism of phenolic compounds that have influence upon the health beneficial value of oats and their products were also observed.ConclusionsAlthough the developed method has broad applicability as a rapid screening method or a rapid metabolite profiling method and in this study has provided valuable metabolic insights, it still must be considered that much greater confidence in metabolite identification, as well as quantitative precision, will be gained by the application of higher resolution chromatography methods, although at a large expense to sample throughput. Follow up studies will apply higher resolution GC (gas chromatography) and LC (reversed phase and HILIC) approaches, oats will be also analysed from across multiple growth locations and growth seasons, effectively providing a cross validation for the results obtained within this preliminary study. It will also be fascinating to perform more controlled experiments with sampling of green tissues, as well as oat grains, throughout the plants and grains development, to reveal greater insight of carbon and nitrogen metabolism balance, as well as resource partitioning into lipid and secondary metabolism.

Drug stability is closely related to drug safety and needs to be considered in the process of drug production, package and storage. To investigate the stability of epalrestat, a carboxylic acid derivative, a reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed in this study and applied to analyzing the degradation kinetics of epalrestat in aqueous solutions in various conditions, such as different pH, temperatures, ionic strengths, oxidation and irradiation. The calibration curve was A = 1.6 × 105C–1.3 × 103 (r = 0.999) with the liner range of 0.5–24 μg/mL, the intra-day and inter-day precision was less than 2.0%, as was the repeatibility. The average accuracy for different concentrations was more than 98.5%, indicating that perfect recoveries were achieved. Degradation kinetic parameters such as degradation rate constants (k), activation energy (Ea) and shelf life (t0.9) under different conditions were calculated and discussed. The results indicated that the degradation behavior of epalrestat was pH-dependent and the stability of epalrestat decreased with the rised irradiation and ionic strength; however, it was more stable in neutral and alkaline conditions as well as lower temperatures. The results showed that the degradation kinetics of epalrestat followed first-order reaction kinetics. Furthermore, the degradation products of epalrestat under stress conditions were identified by UHPLC-PDA-MS/MS, with seven degradation products being detected and four of them being tentatively identified.