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Linusorbs (LOs), significantly influence oil quality and sensory properties of flaxseed oil. Trp-containing LOs exhibit distinct oxidative behavior when γ-tocopherol (γ-T) is present. Polar fractions of crude flaxseed oil were stripped via silica absorption, and reintroduced (LO and γ-T) separately into the oil matrix to investigate their interaction during storage. Compared with crude oil, LOs account for 18.49% reduction of p-anisidine value, while LOs with γ-T contributed to most of the endogenous antioxidant effect in crude oil. γ-T was found to suppress oxidation of Trp-containing LO at early stage (Met form), while facilitate oxidation while at their mid-stage (MetO form, Methionine sulfoxide). In vitro oxidation shows that CLD more likely cleaved into peptide fragments, while few products retain intact ring structures. LC-MS/MS analysis and silicon simulation revealed proximity between MetO and Trp residues, facilitating inter- or intra-molecular reactions and ring structure rupture. Remarkably, the presence of γ-T facilitate these phenomena., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Linusorbs (LOs, cyclolinopeptides) are a class of naturally occurring cyclic hydrophobic peptides found in flaxseed oil, whose oxidation states indicate the oxidative stability and bitterness of flaxseed oil. Subjected to 63 °C accelerated oxidation, most Met-containing LOs in cold-pressed flaxseed oil entirely depleted by the 6th day except CLP, and MetO 2 -containing LOs became the dominant ones. However, no MetO 2 form of Trp-containing LOs, such as CLD, CLF and CLG, were detected. Given their oxidative kinetics, methionine sulfoxide (MetO) residue in some LOs was less sensitive toward oxidation in the presence of Trp (Tryptophan) group, and the oxidative stability of Met-containing LOs was CLP < CLB < CLL ≈ CLM < CLO, as compared to MetO-containing LOs: CLD < CLE < CLC < CLF ≈ CLG. When antioxidant was added into cold-pressed flaxseed oil to assess the additives' antioxidant effect, no significant difference was observed on oil oxidative indices in early stage except α-tocopherol, where they vary dramatically in suppressing Met oxidation of LOs: L-AP (L-ascorbyl palmitate) > TBHQ (tert-butyl hydroquinone) > γ-tocopherol > carnosic acid > α-tocopherol. Besides its ability to suppress oxidation of Trp-containing LOs, L-AP also exhibits superior antioxidant effect on non-Trp-containing LOs due to its amphiphilic property. Due to the prooxidative effects of both α- and γ-tocopherol on LOs that contain Trp, it has been suggested that tocopherols may repair Trp residue on LOs, leading to increased tendency of MetO residues to oxidize. The findings of this research are critical for elucidating the antioxidative mechanism of LOs, which can further lead to the establishment of strategies in suppressing bitter after taste to produce high-quality flaxseed oil., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Linusorbs, known as cyclolinopeptides, are a group of cyclic hydrophobic peptides derived from flaxseed oil with various health benefits. However, the current research efforts on both the biological activities and antioxidant capacities of linusorbs are limited because of existing issues with their purification and characterization. A practical method based on preparative HPLC for isolating 12 linusorbs simultaneously was developed and factors such as the solvent selection, gradient elution program, flow rate, loaded mass, and loading concentration, were optimized. The optimum conditions were an initial acetonitrile (ACN) to water ratio of 40%, final ACN ratio of 80%, eluting time of 21 min, a flow rate of 16 mL/min, sample load of 12.5 mg, and concentration of 80 mg/mL (in methanol). The 12 linusorbs obtained were verified using off-line MS/MS, recording purities of above 95.5%. The method could serve as a practical and fast isolation method enabling further investigation of minor linusorbs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Linusorbs (LOs) are natural peptides found in flaxseed oil that exert various biological activities. Of LOs, LOB3 ([1-9-NαC]-linusorb B3) was reported to have antioxidative and anti-inflammatory activities; however, its anti-cancer activity has been poorly understood. Therefore, this study investigated the anti-cancer effect of LOB3 and its underlying mechanism in glioblastoma cells. LOB3 induced apoptosis and suppressed the proliferation of C6 cells by inhibiting the expression of anti-apoptotic genes, B cell lymphoma 2 (Bcl-2) and p53, as well as promoting the activation of pro-apoptotic caspases, caspase-3 and -9. LOB3 also retarded the migration of C6 cells, which was achieved by suppressing the formation of the actin cytoskeleton critical for the progression, invasion, and metastasis of cancer. Moreover, LOB3 inhibited the activation of the proto-oncogene, Src, and the downstream effector, signal transducer and activator of transcription 3 (STAT3), in C6 cells. Taken together, these results suggest that LOB3 plays an anti-cancer role by inducing apoptosis and inhibiting the migration of C6 cells through the regulation of apoptosis-related molecules, actin polymerization, and proto-oncogenes.

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 β/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.

Flaxseed is becoming increasingly popular as a superfood due to its many health benefits. While flaxseed is considered an oilseed, flaxseed meal (the by-product of flaxseed oil extraction) also contains many nutritional compounds not found in the oil. This study explored the use of a Canadian flaxseed (Linum usitatissimum L.) meal product to fortify bakery foods and improve their nutritional properties. Muffins were made using a control recipe as well as four different formulations that included varying amounts of a standardized flaxseed meal supplement called XanFlax (5, 10, 20, and 40%). The physicochemical properties of the muffins, including their texture, color, sugar content, pH, specific gravity, loss rate, and moisture, were evaluated. Additionally, the sensory attributes contributing to muffin quality were thoroughly examined. The lightness (L*) and yellowness (b*) of the muffins, which were highest in the control group at 82.22 and 34.69, respectively, decreased as the amount of XanFlax increased (p < 0.05). Additionally, the redness (a*) of the muffins increased as the amount of XanFlax increased (p < 0.05). The muffins' sugar content (2.00 brix%) remained consistent across all treatments and controls except for those prepared with 20% XanFlax (2.17 brix%). As the amount of XanFlax powder increased, the pH of the muffins increased significantly. The moisture content in the muffins was highest at 23.71 ± 0.79% in the 10% XanFlax treatment and lowest at 22.06 ± 0.30% in the 40% XanFlax treatment. The muffins enriched with 5% XanFlax had an average height of 5.35 cm and volume of 131.33 mL, surpassing the results for the muffins made with other formulas (p < 0.05). Additionally, the cohesiveness and gumminess of the muffins tended to increase with the addition of XanFlax. The most favorable attributes, namely the appearance, flavor, taste, texture, and overall acceptance, were consistently associated with the 5% and 10% XanFlax treatments (p < 0.05). This study marks the first time a standardized flaxseed gum product, XanFlax, has been described in a functional baking application. [ABSTRACT FROM AUTHOR]

Linosorbs (Los) are cyclic peptides from flaxseed oil composed of the LO mixture (LOMIX). The activity of LO has been reported as being anti-cancer and anti-inflammatory. However, the study of skin protection has still not proceeded. In particular, there are poorly understood mechanisms of melanogenesis to LO. Therefore, we investigated the anti-melanogenesis effects of LOMIX and LO, and its activity was examined in mouse melanoma cell lines. The treatment of LOMIX (50 and 100 μg/mL) and LO (6.25–50 μM) suppressed melanin secretion and synthesis, which were 3-fold increased, in a dose-dependent manner, up to 95%. In particular, [1–9-NαC]-linusorb B3 (LO1) and [1-9-NαC]-linusorb B2 (LO2) treatment (12.5 and 25 μM) highly suppressed the synthesis of melanin in B16F10 cell lines up to 90%, without toxicity. LOMIX and LOs decreased the 2- or 3-fold increased mRNA levels, including the microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) at the highest concentration (25 μM). Moreover, the treatment of 25 μM LO1 and LO2 inhibited the expression of MITF and phosphorylation of upper regulatory proteins such as CREB and PKA. Taken together, these results suggested that LOMIX and its individual LO could inhibit melanin synthesis via downregulating the CREB-dependent signaling pathways, and it could be used for novel therapeutic materials in hyperpigmentation. [ABSTRACT FROM AUTHOR]

Although flax (Linum usitatissimum L.) has long been used as Ayurvedic medicine, its anti-inflammatory role is still unclear. Therefore, we aimed to investigate the anti-inflammatory role of a linusorb mixture (LOMIX) recovered from flaxseed oil. Effects of LOMIX on inflammation and its mechanism of action were examined using several in vitro assays (i.e., NO production, real-time PCR analysis, luciferase-reporter assay, Western blot analysis, and kinase assay) and in vivo analysis with animal inflammation models as well as acute toxicity test. Results: LOMIX inhibited NO production, cell shape change, and inflammatory gene expression in stimulated RAW264.7 cells through direct targeting of Src and Syk in the NF-κB pathway. In vivo study further showed that LOMIX alleviated symptoms of gastritis, colitis, and hepatitis in murine model systems. In accordance with in vitro results, the in vivo anti-inflammatory effects were mediated by inhibition of Src and Syk. LOMIX was neither cytotoxic nor did it cause acute toxicity in mice. In addition, it was found that LOB3, LOB2, and LOA2 are active components included in LOMIX, as assessed by NO assay. These in vitro and in vivo results suggest that LOMIX exerts an anti-inflammatory effect by inhibiting the inflammatory responses of macrophages and ameliorating symptoms of inflammatory diseases without acute toxicity and is a promising anti-inflammatory medication for inflammatory diseases. [ABSTRACT FROM AUTHOR]

Linusorbs (LO), cyclolinopeptides, are a group of cyclic hydrophobic peptides and considered a valuable by-product of flaxseed oil due to numerous health benefits. Currently applied acetone or methanol extraction could contaminate the feedstocks for further food-grade application. Using flaxseed cake as feedstock, this study established a practical method for preparing LO from pressed cake. Firstly, LO composition of 15 flaxseed cultivars was analyzed. Next, cold-pressed cake was milled and screened mechanically. The kernel and hull fractions were separated based on the disparity of their mechanical strength. Monitored by hyperspectral fluorescence, the LO-enriched kernel fraction separated from cold-pressed flaxseed cake was further used as feedstock for LO production. After ethanol extraction, partition, and precipitation, LOs were extracted from cold-pressed flaxseed cake with a purity of 91.4%. The proposed method could serve as feasible flaxseed cake valorization strategy and enable the preparation of other polar compounds such as flax lignan and mucilage. [Display omitted] • Linusorb is prepared from flaxseed cake via dry-screening and ethanol pre-extraction. • Ethanol is proved effective as better substitute for acetone in pre-extraction • Impurities introduced by ethanol could be removed via aqueous precipitation [ABSTRACT FROM AUTHOR]

Soybean oil (SO), recognized for its nutrients and affordability, often oxidizes, leading to nutrient degradation. This study presents a methodology for tailor oil blends to meet specific requirements, using SO blended with corn oil (CO), sunflower oil (SFO), and flaxseed oil (FSO). We analyzed the physicochemical properties, nutrient composition, and antioxidant capacity of various blends. Pearson's correlation analysis was utilized to examine the interrelation between nutrient composition and antioxidant capacity. The principal component analysis identified suitable blend ratios based on the desired nutrient composition and antioxidant capacity. Blend F3 (60% SO, 15% CO, 16% SFO, 9% FSO) demonstrated superior qualities, including a higher α-tocopherol content (138.8 mg/kg), and enhanced antioxidant capacities (DPPH polar: 12 μmol TE/100g, ABTS: 67.3 μmol TE/100g, FRAP: 112 μmol TE/100g), alongside a lower acid value (0.15 mg/g) compared to other blends. We highlight the roles of squalene, γ-tocopherol, δ-tocopherol, and fatty acid composition in enhancing the antioxidant capacity of oil blends. Specifically, the blend labeled F3 emerged as a nutritionally superior and highly antioxidative option among soybean oil blends. This study provides a framework for tailoring oil blends to meet specific nutritional and antioxidative requirements, providing a more flexible and purpose-driven approach to oil blending. • Innovative oil blending boosts nutritional quality. • Enhanced antioxidant capacity through natural methods. • The synergy of α-tocopherol, squalene, and fatty acids in blends boosts health benefits and stability. [ABSTRACT FROM AUTHOR]

Bioactive orbitides (linusorbs, LOs) from flaxseed (Linum usitatisssimum L.) contribute to the bitter flavor of flaxseed oil. Methods were developed for the removal of the bitter-flavored [1–8-NαC], [1-(R s , S s)-MetO]-linusorb B1 (15) from flaxseed oil and now the extracted LOs can be used in nutraceuticals, and even in drug synthesis. The LOs have potential therapeutic applications and may be used as scaffolds that can be modified for increased utility. This review summarizes the current state of knowledge of LOs with regard to their structure and biological activities. Approaches of isolation, structure determination and chemical modification are also reviewed towards the goal of a variety of applications. The availability of naturally isolated LOs in sufficient quantity for chemical modification will allow the conduct of biological assays with the native compound and its analogues. Due to their biological activity, these compounds have the potential for use in health. The modification of the LOs was necessary in order to study the relationship between orbitide structure and biological function. In addition, native and modified LOs can be used as ligands for production of fluorescent tags, affinity purification columns and antibodies. Analogues of natural products can also be used to probe structure-activity relationship studies and the importance of individual amino acids and functional groups in biological activity. Discoveries could lead to value-added flax products that benefit flax growers and consumers. • Summary of the distribution of orbitides (linusorbs, LOs) in flaxseed. • Summary of the quantification of LOs in different varieties of flaxseed. • A systematic nomenclature of 39 LOs is proposed. • A new branch of LO modification through sulfur chemistry has been developed. • Summary of potential physical, chemical, and pharmaceutical applications of LOs. [ABSTRACT FROM AUTHOR]

• Cyanogenic glycosides were successfully removed from whole flaxseed after 72 h of fermentation. • Nutritional content in whole flaxseed were maintained after the fermentation. • Fermentation using lactic acid bacteria can readily be utilized in food production. Flaxseed is categorized as a functional food due to its abundance in oil, α-linolenic acid, dietary fibre, and lignan. However, flaxseed contains cyanogenic glycosides (CGs). Ingestion of CGs can influence nutrient absorption and induce adverse health effects. Due to the presence of CGs in flaxseed many countries prohibit the import and sale of flaxseed and flaxseed-based foods. In this study, whole flaxseed was fermented with a mixed culture of Lactobacillaceae (i.e., Lactobacillus sp., Limosilactobacillus sp., and Lactiplantibacillus sp.) and the concentration of CGs was determined. This process succeeded in completely removing CGs within 72 h in both bench-scale and scale-up studies. In addition, fatty acid composition in flaxseed remained unchanged and concentrations of flaxseed oil, and SDG in flaxseed were increased after fermentation. CG-free flaxseed products are beneficial, as they can be sold as health product ingredients, or as animal feed in markets that currently restrict the use of materials that contain CGs. [ABSTRACT FROM AUTHOR]

A facile strategy for dehulling of six Canadian flaxseed cultivars using ethanol wash in combination with wet-screening was reported. Quantitative determinations of nutritional components including oil, sterol, protein, carbon and cyclolinopide in each separated fraction were also performed. Results showed that an efficient separation of hull, kernel and oil from the whole seed is achieved, because ethanol wash can eliminate the stubborn interference of flaxseed oil, thus facilitating the shell and kernel separation procedure. Among six flaxseed cultivars, the separated hull, kernel and oil account for 20.52–26.66%, 36.02–43.23% and 29.82–32.00% of whole seed, respectively. Triglyceride and sterol contents in separated hull, kernel, oil, whole seed are 2.13–3.34%, 24.68–27.46%, 97.26–98.84%, 37.70–45.82% and 0.02–0.11%, 0.04–0.08%, 0.28–0.53%, 0.11–0.23%, respectively. Besides, flaxseed oil is rich in cyclolinopepides, including new orbitides Q, R and S that have never been reported in these flaxseed varieties. Protein and carbon contents in separated hull, kernel and whole seed are 19.01–26.46%, 34.91–43.80%, 20.15–24.65% and 55.39–56.59%, 45.11–45.93%, 49.61–50.94%, respectively. In addition, the milled flaxseed hull shows a concentration-dependent intrinsic viscosity. This study presents a convenient method for flaxseed dehulling and preliminarily reveals the distribution of different nutrient elements in different parts of flaxseed. • A wet-screening strategy for facilely flaxseed dehulling was reported. • Flaxseed is rich in triglyceride, sterol, fiber, protein, and cyclodipeptides. • Enrichments of different nutrients into corresponding subfractions were achieved. [ABSTRACT FROM AUTHOR]