Glycine-containing flaxseed orbitides.

Five new orbitides, cyclolinopeptides 21-25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z 1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H](+) corresponded to the molecular formulae C59H89N10O10, C58H87N10O10S, C58H87N10O11S, C53H80N9O9S, and C53H80N9O10S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1-10-NαC]-GILVPPFFLI, [1-10-NαC]-GMLIPPFFVI, [1-10-NαC]-GOLIPPFFVI, [1-9-NαC]-GMLVFPLFI, and [1-9-NαC]-GOLVFPLFI for cyclolinopeptides 21-25, respectively. Previously reported orbitides, [1-9-NαC]-ILVPPFFLI (1), [1-9-NαC]-MLIPPFFVI (2), [1-9-NαC]-OLIPPFFVI (3), [1-8-NαC]-MLVFPLFI (7), and [1-8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24 also produced orbitides 1, 2, and 7 by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25 contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23 was isolated for characterization via 1D ((1)H and (13)C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.

Evaluating the cytotoxicity of flaxseed orbitides for potential cancer treatment.

Flaxseed as well as its oil component possess antitumor activities against different types of cancer and have been used by some patients as complementary and/or alternative medicine. Linoorbitides (LOBs) are one family of flaxseed compounds that has implications for anticancer and antioxidant activity. The cytotoxicity of [1-9-NαC]-linusorb-B3 (LOB3), [1-9-NαC]-linusorb-B2 (LOB2), [1-9-NαC],[1-Rs,Ss-MetO]-linusorb-B2 ([MetO]-LOB2) and [1-8-NαC],[1-Rs,Ss-MetO]-linusorb-B1 ([MetO]-LOB1) was measured against human breast cancer Sk-Br-3 and MCF7 cell lines and melanoma A375 cell line. Overall cytotoxicity is cell-type specific. It scales as the hydrophobicity and concentration of the LOBs with the most abundant LOB3 being the most cytotoxic. Oral administration of LOB3 as a potential therapeutic agent might not be applicable as a much too high and/or frequent dose would be required to achieve a serum concentration of 400-500 µg/mL due to bioavailability and pharmacokinetic factors. However, LOB3 may be suitable for topical treatment formulations or as a lead compound in developing anticancer LOB derivatives.

Simulated moving bed purification of flaxseed oil orbitides: unprecedented separation of cyclolinopeptides C and E.

The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated.

New flaxseed orbitides: Detection, sequencing, and (15)N incorporation.

Three new orbitides (cyclolinopeptides 17, 18, and 19) were identified in flaxseed (Linum usitatissimum L.) extracts without any form of purification. Their structures were elucidated by a combination of (15) N-labeling experiments and extensive tandem mass spectrometry (MS/MS) with electrospray ionization (ESI). Putative linear peptide sequences of the new orbitides were used as the query in the Basic Local Alignment Search Tool (BLAST) searches of flax genome database. These searches returned linear sequences for the putative precursors of cyclolinopeptides 17 and 19 among others. Cyclolinopeptide 18 contains MetO (O) and is not directly encoded, but is a product of post-translation modification of the Met present in 17. The identification of precursor proteins in flax mRNA transcripts and DNA sequences confirmed the occurrence and amino acid sequences of these orbitides as [1-9-NαC]-MLKPFFFWI, [1-9-NαC]-OLKPFFFWI, and [1-9-NαC]-GIPPFWLTL for cyclolinopeptides 17, 18, and 19, respectively.

Effect of cyclolinopeptides on the oxidative stability of flaxseed oil.

Polar compounds present in flaxseed oil increase its oxidative stability. Flaxseed oil becomes less stable to oxidation when filtered with silica. This observation may be linked to antioxidant compounds present in flaxseed oil. Flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. The polar fractions from flaxseed oil were then added back to silica-treated flaxseed oil to determine the impact of fractions containing polar compounds on oxidative stability (induction time) at 100 °C. A polar fraction containing mainly cyclolinopeptide A (CLA, 1), but also containing ß/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.36 ± 0.28 to 3.20 ± 0.41 h. When oxidative stability was determined immediately after addition of the polar fractions other flaxseed fractions and solvent controls did not affect oil stability. However, when the oxidative stability index (OSI) test was delayed for three days and oil samples were held at room temperature after the addition of the polar fractions to the flaxseed oil, it was observed that the control oil treated with silica had become highly sensitive to oxidation. A polar fraction containing a mixture of CLs (1, 5, 7, 9, 11), improved the oxidative stability of peptide-free oil with respect to the control when the OSI measurement was made three days after adding the fraction. In addition, effects of 1 on the oxidative stability of peptide-free oil containing divalent metal cations was investigated.

Detection, isolation and characterisation of cyclolinopeptides J and K in ageing flax.

Methionine sulfone containing peptides CLs J (11) and K (12) may be produced from their reduced forms by oxidation but it is not known if these compounds occur in foods that contain flax. These compounds have been reported to possess greater immunosuppressive activity than their reduced methionine sulfoxide peptide forms 4 and 6, respectively. Since 11 and 12 have not been detected in commercial flax oil and milled flax seed, we tested for their presence in flax food products. Here we report that 11 and 12 accumulate in ground flaxseed that is exposed to air and heat (100°C) for more than 4h. Standards of 11 and 12 were prepared, isolated and extensively characterised using HPLC-MS/MS, 1D and 2D NMR methods. We also report the excellent thermal and oxidative stability of these peptides. Due to the harsh conditions required to produce 11 and 12, it is expected that their levels in flax based foods would be low and therefore their presence could serve as an indicative measure of severe oxidation of a food product.

Rapid reversed-phase liquid chromatography separation of cyclolinopeptides with monolithic and microparticulate columns.

Three monolithic C(18)-bonded silica gel columns i.e. Chromolith SpeedROD (CSR), Chromolith Performance (CP), and Chromolith High Resolution (CHR), MerckKGaA Darmstadt, Germany and two particle-based columns i.e. ZORBAX Eclipse XDB-C(18) (ZEX), Agilent and POROS R1/20 (POR), Applied Biosystems were compared for their performance in separating a mixture of flaxseed cyclolinopeptides (CLs). Gradient mobile phases of acetonitrile and water were optimized for each column. The performance of CHR column in profiling CL standards, measured as the resolution of individual CL, selectivity, and peak asymmetry exceeded the performance of traditional particle-packed columns and the other monolithic columns. The profiling of CLs in aqueous methanolic flaxseed extract was optimized for high-throughput analysis. A total analysis time of 1.5 min at a flow rate of 3.0mLmin(-1) was achieved on a CSR column. Injection of over 2000 methanol extracts of flaxseed on a CSR column had no impact on backpressure or resolution of a standard CL mixture.

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: de novo incorporation of deuterated tryptophans and quasi-natural compounds.

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1'-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to "quasi-natural" products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1'-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1'-methylindolyl-3'-acetaldoxime and 1'-methylindolyl-3'-acetothiohydroxamic acid (quasi-natural products) into 1'-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.