Anthocyanins from red cabbage are of great importance for their applications in the food industry as natural colorants and their beneficial effects on human wellness as natural antioxidants. This study aimed to develop an effective method for the isolation of anthocyanins with the help of a combination of alternate recycling and direct recycling preparative liquid chromatography. Ten major components of anthocyanins from red cabbage were isolated and their structures were identified by HPLC-MS/MS. Meanwhile, the stability of the isolated anthocyanins under various light conditions was also investigated so as to provide data for their storage. In sum, the results showed that twin column recycling preparative chromatography is an effective method for the isolation of anthocyanin monomers with similar structures. Besides, the stability of various anthocyanins from red cabbage was related to the number of acylated groups and mainly affected by illumination.
Anthocyanins and PAs are the two most common flavonoids, which are widely present among diverse species. Great progress has been made in their synthesis and regulation. In this study, we analyzed the metabolic fluxes from their synthetic precursor leucoanthocyanins, which were obtained by overexpression of dihydroflavonol 4-reductase (DFR) in vitro and in vivo. The unstable product leucocyanidin generated in the CsDFRa enzymatic reaction was easily converted into C-type carbocations under weak acidic conditions, which could be further involved in the synthesis of C-type PAs in vitro. Additionally, the metabolites in tobacco overexpressing CsDFRa and Arabidopsis thaliana DFR and anthocyanidin synthase (ANS) mutants were investigated. In CsDFRa transgenic tobacco, the content of anthocyanins in the petals was greatly increased, but no catechin or PA was detected. In A. thaliana, EC-type carbocation was mainly accumulated in the wild type (WT), and the C-type carbocation was only detected in the ans mutant. In tea plant, the accumulation of C-type PAs is strong positively correlated with the expression of CsDFRa. In summary, leucocyanidin is not only involved in the synthesis of downstream anthocyanin and epicatechin but also can be converted into C-type carbocation to participate in the synthesis of C-type PAs. Hence, from leucocyanidin, three metabolic fluxes were formed toward catechin, cyanidin, and C-type carbocation. These results enriched the metabolic fluxes of leucoanthocyanins and further elaborated the roles of DFR in the process of C-type PA formation.
Introduction: Ambient pressure electrospray ionisation ion mobility spectrometry coupled to high-performance liquid chromatography (HPLC) was used to detect alkaloids from different parts of Sophora alopecuroides L. extracts. Multiplexing ion mobility spectrometry (IMS) was used to improve the signal-to-noise ratio while maintaining high resolving power for the detecting of eluents from HPLC separation. Material and methods:The alkaloids profile and distribution are demonstrated by retention time-drift time two-dimensional spaces, and the contents of five major alkaloids including sophoridine, sophocarpine, cytisine, aloperine, and matrine were determined in the leaf, skin, stem, seed kernel, and seed husk using the HPLC-IMS method. This method offers extra separation ability to isomers such as matrine and sophocarpine, which can be difficult to distinguish by mass spectrometry. Results:The reduced mobilities for cytisine, sophoridine, sophocarpine, matrine, and aloperine are 0.828, 0.718, 0.731, 0.725, and 0.769 cm 2 /V/s, respectively. The limits of detection are 0.553, 0.488, 0.479, 0.484, and 0.513 ug/mL. This method adds extra separation ability to HPLC to resolve co-eluted peaks and provides another qualitative parameter besides HPLC retention time.
In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two‐dimensional separation. To improve the performance of hyphenated CE‐IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5–250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE‐ESI‐IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra‐violet transparent compounds or none fluorescent compounds.
Brassinosteroid (BR), a kind of polyhydroxylated steroid hormone, plays an important role in physiological and biochemical processes in plants. Studies were mainly focused on BR signaling and its exogenous spraying to help enhance crop yields. Few research studies are centered on the accumulation pattern of BR and its mechanism. Yet, it is crucial to unlock the mystery of the function of BR and its cross action with other hormones. Tea (Camellia sinensis (L.) O. Kuntze) is one of the important economic crops in some countries, and new shoots are the raw materials for the preparation of various tea products. Different concentrations of exogenous BR were reported to have different effects on growth and development. New shoots of tea plants can thus be considered a valuable research object to study the accumulation pattern of BR. In this study, the quantity of five BR components (brassinolide, 28-norbrassinolide, 28-homobrassinolide, castasterone, and 28-norcastasterone) in different tissues of tea plants, including buds (Bud), different maturity of leaves (L1, L2), and stems (S1, S2) were determined by UPLC-MS/MS. A total of 15 cDNA libraries of the same tissue with three repetitions for each were constructed and sequenced. The BR-accumulation pattern and gene expression pattern were combined together for weighted gene co-expression network analysis (WGCNA). BR-accumulation-relative genes were then screened using two methods, based on the K.in value and BR biosynthetic pathway (ko00905), respectively. The result showed that photosynthesis-related genes and CYP450 family genes were actively involved and might play important roles in BR accumulation and/or its accumulation pattern. First and foremost, feedback inhibition was more likely to dominate the accumulation pattern of BR in the new shoots of tea plants. Moreover, three conserved miRNAs with their target transcriptional factors and target mRNAs had been figured out from negative correlation modules that might be strongly linked to the BR-accumulation pattern. Our study provided an experimental basis for the role of BR in tea plants. The excavation of genes related to the accumulation pattern of BR provided the possibility of cross-action studies on the regulation of BR biosynthesis and the study between BR and other hormones.
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