Antioxidant Activity and Oxidative Stability of Flaxseed and Its Processed Products: A Review

Abstract

Flaxseed (Linum usitatissimum) is one of the most important crops worldwide due to its nutritional and functional properties. Given the diversity of flax and its processed products, this review aimed to systematize and analyze data on their antioxidant properties, oxidative stability, and content of biologically active substances. The literature search was conducted using the following databases: Scopus and The Lens. This review examines the approaches to studying the antioxidant properties, oxidative stability, and content of biologically active substances of flax and its processed products, which are used in the food industry, highlighting the advantages and limitations of the methods employed. For the analysis of AOA and OS in flaxseeds and their processing products, the most common approach is the in vitro model. For AOA assessment, non-standardized methods such as DPPH^•, FRAP, and ABTS^•+ are most frequently used, while standard methods for determining OS (PV, AV, p-AnV, CDs, CTs, TBARSs, OSI) are employed. However, these parameters are integral and cannot fully explain the underlying processes. In our opinion, the most promising directions for further research are the standardization of methods for analyzing the antioxidant activity (AOA) of flaxseed and its processing products. Furthermore, expanding the methodological framework will lead to a better understanding of the mechanisms of oxidative processes and how to inhibit them. An expanded set of AOA assessment methods will allow researchers not only to study the action of antioxidants but also to predict it. This is particularly relevant since the same antioxidant can exhibit both antioxidant and pro-oxidant effects.

1. Introduction

Flax (Linum usitatissimum) belongs to the family Linaceae, genus Linum, and is an annual crop that produces small oval seeds with pointed tips, varying in color from golden yellow to reddish brown [1,2]. The flaxseed coat consists of the hull, the thin endosperm, and two embryos [3], yet every part of the flax plant can be used commercially. Thus, flaxseed is an important global crop, cultivated primarily for the production of oil, fiber, food, and animal feed [2,3,4]. There are two main types of flax grown worldwide—fiber flax and seed flax. According to FAOSTAT data https://www.fao.org/faostat/en/#data/QCL (date of access 19 September 2025) [5], in 2023, the top five countries for flaxseed cultivation were the Russian Federation (1,100,000 t), Kazakhstan (361,697 t), Canada (272,736 t), China (260,000 t), and India (166,753 t).

Flaxseed contains a large amount of various macro- and micronutrients, as well as biologically active substances. The ratio of the main components of flaxseed may vary depending on the genotype and environment [1,4]. The component composition is crucial for evaluating the nutritional value and suitability for processing of flaxseeds. At present, flax is employed extensively in a variety of industrial areas (Figure 1).

In the food industry, flaxseed and its processed products are used to develop different functional food products. For example, in confectionery products, fermented flaxseed is used in dark chocolate as a source of prebiotics [6]. In bread and bakery products [7,8], including cookies, flaxseeds (including milled seeds, flour, and cake) can be used not only as a source of biologically active substances, but also to regulate the physical, chemical, and rheological properties of dough and final products. Marand et al. showed that the addition of flaxseed powder to yoghurt led to an increase in pH, acidity, water-holding capacity, viscosity, and antioxidant activity, as well as a decrease in saturated fatty acids (SFAs) and an increase in polyunsaturated fatty acids (PUFAs) [9]. Another rapidly developing area is the development of methods for the isolation, identification, evaluation of properties, and application of biologically active substances extracted from flax and its processed products. For example, cyclolinopeptides isolated from flaxseed oil can be used as an additive to control microbial contamination of high-fat foods, as they have an inhibitory effect on Listeria monocytogenes [10]. These studies are just a few examples of the possible uses of flax and flax products in food products. More detailed information on the use of flax and flax products in the food industry can be found in reviews [2,11,12,13,14,15,16].

Flax and its processed products are actively studied in terms of their antioxidant content and ability to inhibit oxidative processes in food products during storage. However, there are currently no approved standardized methods for assessing the antioxidant activity of flax and its processed products. Various in silico, in vitro, and in vivo methods are used to assess antioxidant activity. However, there are currently no reviews devoted to the methods and approaches used to assess antioxidant activity and oxidative stability. The majority of published reviews briefly discuss traditionally used in vitro or in vivo methods, without specifying the advantages and disadvantages of these methods.

Thus, the objective of this review is to systematize and analyze the literature focused on the study of the antioxidant activity and oxidative stability of flaxseeds and their processed products, with an emphasis on the methods and approaches used to identify the advantages and disadvantages of their use.

2. Research Methodology

The literature search was carried out using the following keywords and phrases: “flaxseed” or “linseed” antioxidant activity, oxidative stability of “flaxseed” or “linseed”, biologically active substances of “flaxseed” or “linseed”, methods of “flaxseed oil” or “linseed oil” research, “flaxseed oil” or “linseed oil” antioxidant activity, oxidative stability of “flaxseed oil” or “linseed oil”, biologically active substances of “flaxseed oil” or “linseed oil”, methods of “flaxseed oil” or “linseed oil” research, “flaxseed hull” or “linseed hull” analysis, “flaxseed meal” or “linseed meal” analysis, “flaxseed cake” or “linseed cake” analysis, “flaxseed flour” or “linseed flour” analysis, “methods for assessing biologically active substances of flax”, according to the Scopus and The Lens databases by subject field (article title, abstract, keywords)—update date 5 March 2025.

A search using keywords and phrases revealed over 10,000 articles (from 1950 to 2025). Before the publications were analyzed, all duplicate manuscripts were deleted from the selection. The next principal task was to select original research articles as primary sources. Inclusion and exclusion criteria were used to select publications.

Inclusion criteria were as follows: (1) open access publications, (2) only original research articles, (3) publications in English.

Exclusion criteria were as follows: (1) books, reviews, abstracts, congresses and conferences, systematic reviews, brief reports, (2) duplicate studies, (3) articles whose full text is not available (abstract only).

A total of 156 publications were selected for analysis based on inclusion/exclusion criteria. Although reviews were excluded from the publications for analysis, they were included in the discussion of the findings.

Since flaxseeds and flaxseed products have specific characteristics as research subjects, an individual analysis of approaches and methods for assessing their antioxidant activity and oxidative stability was conducted.

3. Study of Antioxidant Activity and Oxidative Stability of Flaxseeds

Flaxseed is a valuable oilseed crop and a rich source of biologically active substances (dietary fiber, phenolic compounds, proteins, cyclolinopeptides, etc.) [2,17,18,19,20]. Depending on various factors, the composition of flaxseed can vary: protein, 17.3–28%; fats, 30–56.7%; moisture, 3.47–5.4%; fiber, 7.1–30%; ash, 3.6–7% [16,21,22]. It is known that both the variety and origin of the seeds significantly affect the fatty acid composition, phenolic acids, lignans, and, consequently, the antioxidant activity of flax [1,23]. As part of this review, we analyzed publications focused on research into antioxidant activity (AOA), oxidative stability (OS), and the content of biologically active substances (BASs) in flaxseed. Examples of data used to evaluate these parameters are presented in

Table 1. Parameters used to assess the antioxidant activity and content of biologically active substances in flaxseed in vitro.

Sample	Parameters				Reference
	AOA	Value	BAS	Value
flaxseed	DPPH• FRAP	35.68–66.76% 608.95–5031 μM Fe2+/g	TPC TFC	69.34–84.13 mg GA/100 g 2.1–5.11 mg QE/100 g	[21]
flaxseed (4)	ORAC DPPH• FRAP	38.43–88.16 μmol TE/g 3.25–8.40 μmol TE/g 32.22–61.65 μmol TE/g	TPC * SDG SECO α-linolenic acid	235.15–389.65 mg GA/100 g 61.98–144.66 μg/g 76.54–243.69 μg/g 47.44–53.67%	[23]
flaxseed	DPPH• ORAC	0.081–0.134 g/L (EC50) 0.36–1.07 mmol TE/g	SDG SECO	5.12–77.98 mg/g 6.43–15.84 mg/g	[24]
flaxseed	DPPH• FRAP H2O2 NO	39.07 ± 2.84 µg TE/g 64.57 ± 12.09 µg TE/g 14.33 ± 3.86 µg TE/g 57.72 ± 3.58 µg TE/g	TPC TFC	95.18 mg GA/100 g 28.61 mg QE/100 g	[25]
flaxseed	DPPH•	27.5–89.9%	chlorogenic acid methyl gallate gallic acid ellagic acid rutin coffeic acid coumaric acid vanillin cinnamic acid	1932.20 µg/mL 284.31 µg/mL 155.10 µg/mL 120.86 µg/mL 32.81 µg/mL 32.78 µg/mL 17.02 µg/mL 16.45 µg/mL 8.84 µg/mL	[26]
flaxseed	DPPH• TAC	0.25 ± 0.02 µmol TE/mg 0.21 ± 0.04 µmol TE/mg	α-linolenic acid	73.2 ± 0.4%	[27]
flaxseed			TPC SDG	85 ± 5 mg CE/g 333 ± 15 mg/g	[28]
flaxseed	DPPH• ALA	42.2–87.5% 56.7–88.2%	TPC TFC	1360–3260 mg GA/100 g 190–480 mg CE/100 g	[29]
flaxseed	DPPH•	39.47–62.10%	TPC TFC	11.5–15.5 mg/g 1.5–5.0 mg/g	[30]
flaxseed	DPPH•	40–50%	TPC * TFC α-linolenic acid	0.6–1.67 mg GA/100 g ~0.40–1.08 mg QE/g 52.40%	[31]
flaxseed (32)	DPPH• FRAP ABTS•+	32.56–46.22 mg TE/100 g 0.58–1.08 mg TE/g 14.22–36.14 mmol TE/g	TPC PSC SDG SECO	109.93–246.88 mg/100 g 56.52–125.12 mg/g 11.37–21.25 mg/g 0.76–3.16 mg/g	[32]
flaxseed (5)	DPPH•	9.42–13.14 mmol/kg	CaC TPC TFC α-linolenic acid	0.14–0.66 μg/g 178.81–243.73 mg/100 g 255.71–424.29 mg/100 g 51.19–56.51%	[33]
flaxseed	ABTS•+ DPPH• FRAP ORAC	0.90 mmol TE/g 0.12 mmol TE/g 0.13 mmol TE/g 7.6 µmol TE/g	TPC TFC α-linolenic acid	1538.7 µg/g 1.7 mg/g 60.08%	[34]
flaxseed non-defatted	DPPH• FRAP	25.7–76.3% 0.062 ± 0.007 mmol TE/g	TPC	61.3 ± 0.02 mg CE/100 g	[35]
flaxseed defatted	DPPH• FRAP	19.7–76.1% 0.058 ± 0.009 mmol TE/g	TPC	98.8 ± 0.01 mg CE/100 g	[35]
flaxseed (5)	DPPH• ABTS•+	10.20–13.80 μmol TE/g 10.50–12.99 μmol TE/g	TPC *	3.8–4.56 mg GA/100 g	[36]
flaxseed	DPPH•	21.68–71.24%	TPC	483–3315 mg GA/100 g	[37]
flaxseed (2)	ABTS•+ DPPH• FRAP	3.38–3.70 mmol TE/g 1.16–1.56 mmol TE/g 0.33–0.76 mmol TE/g	α-linolenic acid	396.56–544.85 mg/g	[38]
flaxseed (8)	DPPH• ALA	63.06–86.58% 65.59–85.29%	TPC TFC SDG SECO	2560–3286 mg GA/100 g 232.53–346.67 mg CE/100 g 14.78–124.27 mg/100 g 396.49–1518.2 mg/100 g	[39]
flaxseed (3)	ABTS•+ DPPH• FRAP CA	0.46–5.06 mmol TE/g 0.06–0.51 g/L (EC50) 0.44–4.96 mmol FeS04/g 0.11–6.54 g/L (EC50)	SDG SECO	9.55–107.37 mg/g 2.11–21.74 mg/g	[40]
flaxseed (15)	DPPH• ABTS•+ CA CUPRAC	1.89–6.03 µg/mL (IC50) 0.61–3.21 µg/mL (IC50) 0.92–3.84 µg/mL (IC50) 6.34–11.78 µmol TE/mg	TPC TFC α-linolenic acid	613.6–3164.6 mg GA/g 176.25–689.20 mg QE/g 39.21–54.2%	[41]
flaxseed	DPPH• FRAP	57.2 ± 11.0 mg TE/100 g 8.5 ± 0.4 mmol FeS04/100 g	TPC TFC α-linolenic acid	142.5 ± 7.2 mg GA/100 g 53.9 ± 2.4 mg epicatechin/100 g 56.75 ± 0.19%	[42]

* The table presents the total content of the determined compound classes. Additionally, the authors of the studies also analyzed the profile of the corresponding compounds. ABTS^•+—radical 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate); ALA—antioxidant activity in linoleic acid; AOA—antioxidant activity; BAS—biologically active substance; CaC—carotenoid content; CA—chelating activity; CE—catechin; CUPRAC—cupric ion (Cu²⁺) reducing power assay; DPPH^•—radical 1,1-diphenyl-2-picrylhydrazyl; FRAP—ferric reducing antioxidant power; GA—gallic acid; ORAC—oxygen radical absorbance capacity; PSC—phytosterol content; QE—quercetin; SDG—secoisolariciresinol diglucoside; SECO—secoisolariciresinol; TAC—total antioxidant capacity; TE—trolox; TFC—total flavonoid content; TPC—total phenolic content.

.

In terms of studying the AOA of flaxseed, most research is focused on analyzing the content of BASs and their correlation with AOA (Table 1). Although flaxseed is a rich source of BASs, comparative studies on the compositional profile of different flax cultivars [1,23,32,33,36,38,39,40,41] and their AOA are actively being conducted. It is also worth noting separately the investigation into the effect of flaxseed germination on AOA and BAS content [43,44,45,46,47]. In general, all studies, regardless of the flaxseed variety, have noted an improvement in antioxidant activity. At the same time, studies have noted both an increase in the level of individual polyphenolic compounds [43] and a decrease in the total content of polyphenols [44]. Although phenols are the main compounds associated with AOA, an increase in AOA may be associated with an increase in other components such as fresh synthesis of vitamin C, flavonoids, tocopherols, quercetin, etc. [45]. It should be noted that an important factor is the duration of germination, since, for example, on the second day of germination, the total content of polyphenols decreased, relative to unsprouted seeds, by about 2-fold, while on the 10th day, the total content of phenols and flavonoids increased 5.6-fold and 55-fold, respectively [46].

In addition to studies of AOA and the content of BASs in flaxseeds, studies of oxidative stability during storage are also being conducted [47,48,49,50,51,52,53]. To control oxidation processes during storage, the following are used: peroxide value (PV), content of free fatty acids, volatile organic compounds (VOCs), Fourier transform infrared spectroscopy (FTIR), sensory analysis (SA), thiobarbituric acid reactive substances (TBARSs), etc. It has been shown that the oxidation rate depends on the flaxseed variety and storage conditions (temperature, humidity, post-harvest processing, packaging, etc.).

4. Flaxseed Oil: Methods of Production, Antioxidant Activity, and Oxidative Stability

4.1. Methods of Production of Flaxseed Oil

The basic method for obtaining flaxseed oil is single “cold” pressing—FOC, which involves the use of a screw oil press with an outlet oil temperature not exceeding 60 °C [54,55]. However, there are many other ways to obtain oil, including extraction using solvents—FOE [56,57], where hexane is usually used as the solvent, and, less commonly, isopropyl alcohol is used. In some cases, methods such as extraction using supercritical CO₂ (FOSC) [58], Soxhlet extraction (FOSE) [59], double or triple “cold pressing” (FOC) (double or triple) [60], and others are used to obtain oil. Oils are obtained from the seeds themselves (including germinated seeds) (FOCG) [55] and from their components, such as hulls [61]. In a variety of cases, oils are desorbed, i.e., purified from extraneous components using silica gel (FOD) [19,62]. The use of different methods for obtaining flaxseed oil leads to different final component compositions [63], including the content of substances that have antioxidant activity. Differences in the component composition of oil are also associated with the use of various analysis methods [63].

4.2. Investigation of Antioxidant Activity and Oxidative Stability of Flaxseed Oil Under In Vitro Conditions

Studies of the antioxidant activity of flaxseed oil are mainly focused on assessing its changes depending on various factors, such as temperature and storage duration [64], assessing the content of BASs [56,65], and finding correlations between the content of BASs, AOA indicators, and OS [55,66].

Oxidation is a complex process with various oxidation products, the content of which can be assessed using appropriate indices [67]. Specific parameters (indices) and analytical methods can be selected depending on the research objectives. Given the high content of polyunsaturated fatty acids, including omega-3 fatty acids, in flaxseed oil, the study of oxidative stability and antioxidant activity (

Table 2. Parameters used to evaluate the antioxidant activity, oxidative stability, and biologically active substance content in flaxseed oils obtained by various methods.

Sample	Parameters						Reference
	AOA	Value	OS	Value	BAS	Value
FOC (5)	-	-	p-AnV PV AV DSC TOTOX	0.65–0.94 1.21–6.9 meq O2/kg 0.4–1.6 mg KOH/g 37–51 min (120 °C) 3.17–14.74	α-linolenic acid	55.91–63.11%	[54]
FOC	ABTS•+ DPPH• FRAP	17.9 ± 1.1 μmol TE/kg 9 ± 0.2 μmol TE/kg 169.9 ± 12.9 mmol Fe(II)/kg	OSI PV AV	1.6 ± 0.06 h 17.5 ± 0.5 meq O2/kg 3.7 ± 0.2 mg KOH/g	TPC TTC CaC ChC α-linolenic acid	15.4 ± 1.4 mg GA/kg 32.8 ± 1.6 mg/100 g 2.1 ± 0.02 mg/kg 0.61 ± 0.02 mg/kg 56.0%	[55]
FOCG	ABTS•+ DPPH• FRAP	1612.1 ± 45.3 μmol TE/kg 609.9 ± 11.4 μmol TE/kg 3092.2 ± 85 mmol Fe(II)/kg	OSI PV AV	6.2 ± 0.06 h 6 ± 0.4 meq O2/kg 37.2 ± 0.2 mg KOH/g	TPC TTC CaC ChC α-linolenic acid	572.8 ± 13.1 mg GA/kg 43.1 ± 1.5 mg/100 g 15.7 ± 0.02 mg/kg 16.4 ± 0.02 mg/kg 55.0%	[55]
FOE	-	-	PV AV p-AnV OSI TOTOX	0.29 ± 0.10 meq O2/kg 2.16 ± 0.23 mg KOH/g 0.740 ± 0.080 1.28 ± 0.16 h 1.32 ± 0.23	TPC TTC TSC α-linolenic acid	19.5 ± 0.49 mg GA/kg 447 ± 12 mg/kg 3510 ± 2.5 mg/kg 53.4 ± 1.4%	[56]
FOE (polar solvent)	FRAP ABTS•+ DPPH•	10 ± 0.03 µmol/mL 43 ± 0.52% 80 ± 0.34%	AV PV	0.80 ± 0.25 mg KOH/g 0.95 ± 0.19 meq O2/kg	TFC TPC α-linolenic acid	402 ± 0.95 µg CE/mg 1975 ± 1.11 mg GA/100 g 53.29%	[57]
FOE (non- polar solvent)	FRAP ABTS•+ DPPH•	11 ± 0.39 µmol/mL 44 ± 0.42% 82 ± 0.21%	AV PV	0.84 ± 0.14 mg KOH/g 0.99 ± 0.21 meq O2/kg	TFC TPC α-linolenic acid	441 ± 0.87 µg CE/mg 2120 ± 1.07 mg GA/100 g 57.35%	[57]
FOC, FOH (FOE, FOSC)	PCL	0.6–1.2 μm TE/g	DSC	105–163 °C (t onset of oxidation)	SDG	20.19–51.72 mg/g	[58]
FOC	-	-	PV AV	0.85 ± 0.04 meq O2/kg 1.59 ± 0.07 mg KOH/g	Vit E TPC α-linolenic acid	380 mg/kg 118 mg GA/g 49.33–51.01 g/100 g	[59]
FOSE	-	-	PV AV	0.75 ± 0.01 meq O2/kg 0.93 ± 0.03 mg KOH/g	Vit E TPC α-linolenic acid	410 mg/kg 139 mg GA/g 48.14–50.95 g/100 g	[59]
FOC	-	-	PV	2 ± 0.03 meq O2/kg	TPC TFC α-linolenic acid	10 mg GA/100 g 5 mg rutin/100 g 46.03–47.15%	[60]
FOH	DPPH•	64.7%	AV PV p-AnV CDs CTs OSI TOTOX COX	1.5 ± 0.14 mg KOH/g 1.85 ± 0.08 meq O2/kg 1.10 ± 0.17 1.50 ± 0.10 0.24 ± 0.06 1.4 ± 0.28 h 4.8 ± 0.16 11.87 ± 0.20	TFC TPC CaC ChC PLC α-linolenic acid	18 ± 1.40 mg luteolin/100 g 84 ± 9.36 mg GA/100 g 7.82 ± 0.64 mg/kg 3.16 ± 0.28 mg/kg 2.27 ± 0.32% 47.19 ± 0.41%	[61]
FO	TAC DPPH• ALA	65.44 ± 0.39% 45.75 ± 0.42% 28.49 ± 0.72%	OSI PV	3.62 ± 0.02 h 0.23 ± 0.02 meq O2/kg	TSC * TPC * α-linolenic acid	103.6 mg/100 g 145 GA/100 g 54.67 ± 0.07 mg/100 g	[64]
FOC	-	-	PV p-AnV AV CDs CTs	2.04 ± 0.15 meq O2/kg 0.52 ± 0.03 1.49 ± 0.02 mg KOH/g 2.08 ± 0.03 0.02 ± 0.01	ChC TTC CaC TPC TFC α-linolenic acid	6.78 ± 0.01 mg pheophytin/kg 37.00 ± 0.02 mg/100 g 0.06 ± 0.00 mg/100 g 136.93 ± 1.36 mg GA/100 g 18.75 ± 0.36 mg luteolin/100 g 59.34 ± 1.34%	[65]
FOC	DPPH•	1.58 ± 0.17 TE, mM/kg	AV PV p-AnV CDs CTs	0.17 ± 0.07 mg KOH/g 0.60 ± 0.10 meq O2/kg 0.87 ± 0.57 1.94 ± 0.28%E 0.30 ± 0.27%E	α-linolenic acid	51.2 ± 3.7%	[66]
FOC	FRAP DPPH• ABTS•+	78.63 ± 1.64 μmol TE/100 g 185.36 ± 7.62 μmol TE/100 g 1040.86 ± 41.69 μmol TE/100 g	PV p-AnV AV TOTOX COX OSI	0.61 ± 0.02 meq O2/kg 0.39 ± 0.02 0.37 ± 0.01 mg KOH/g 1.61 13.14 4.87 ± 0.21 h	TTC * TPC TSC * α-linolenic acid	44.04 ± 1.04 mg/100 g 2.93 ± 0.20 mg GA/100 g 335 ± 7 mg/100 g 52.12 ± 0.28%	[68]
FO	DPPH•	74.7%	-	-	TPC TFC	32.2 mg GA/100 g 22.82 mg QE/100 g	[69]
FOC (15)	DPPH•	50.1–56.3%	PV AV p-AnV TOTOX OSI COX DSC	1.23–4.50 meq O2/kg 0.53–3.15 mg KOH/g 0.07–1.43 3.11–9.07 2.85–4.96 h 12.03–15.40 56.48–125.19 min	CaC TPC ChC α-linolenic acid	18.34–67.97 mg β-carotene/kg 60.25–115.12 mg ferulic acid/100 g 0.06–3.93 mg pheophytin/kg 44.90–64.62%	[70]
FOC (6)	DPPH•	1.10–2.30 mM TE/kg	AV PV p-AnV CDs CTs	0.52–1.74 mg KOH/g 0–1.17 meq O2/kg 0.27–0.73 1.51–1.89 µmol/g 0.16–0.28 µmol/g	TPC TTC ChC TSC α-linolenic acid	55.8 mg GA/kg 588.7 mg/kg 0.79 mg/kg 5171.7 mg/kg 49.3–59.3%	[71]
FOEG	DPPH•	51.4%	AV p-AnV PV CDs CTs COX	2.48 ± 0.08 mg KOH/g 1.6 ± 0.25 2.4 ± 0.16 meq O2/kg 2.12 ± 0.10 0.44 ± 0.12 10.82 ± 0.32	ChC CaC TFC TPC α-linolenic acid	7.37 ± 0.28 mg/kg 6.27 ± 0.07 mg/kg 11.82 ± 1.20 mg luteolin/100 g 73.11 ± 4.29 mg ferulic acid/100 g 41.07–43.24%	[72]

* The table presents the total content of the determined compound classes. Additionally, the authors of the studies also analyzed the profile of the corresponding compounds. ABTS^•+—radical 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate); ALA—antioxidant activity in linoleic acid; AV—acid value; AOA—antioxidant activity; BAS—biologically active substance; CaC—carotenoid content; CD—conjugated diene; CE—catechin; ChC—chlorophyll content; CoQ—coenzyme Q content; COX—calculated oxidizability value; CT—conjugated triene; DPPH^•—radical 1,1-diphenyl-2-picrylhydrazyl; DSC—differential scanning calorimetry; FRAP—ferric reducing antioxidant power; FO—flaxseed oil; FOC—cold-pressed oil; FOE—oil extracted using solvents; FOCG—cold-pressed oil from germinated seeds; FOEG—oil extracted from germinated seeds; FOH—oil from flaxseed hull (cases where oil is obtained from hulls, but using different approaches, are also indicated as FOH, but the approach is indicated in brackets; for example, extraction—FOE); FOSC—oil extracted by supercritical CO₂; FOSE—Soxhlet extraction of oil; GA—gallic acid; OS—oxidative stability; OSI—oxidative stability index; p-AnV—para-anisidine value; PCL—photochemiluminescence assay; PLC—phospholipid content; PV—peroxide value; QE—quercetin; SDG—secoisolariciresinol diglucoside; TAC—total antioxidant capacity; TE—trolox; TFC—total flavonoid content; TOTOX—total oxidation; TTC—total tocopherol content; TPC—total phenolic content; TSC—total sterol contents; Vit E—vitamin E.

) in this object is of special importance.

As shown in Table 2, an in vitro model using various methods and approaches is widely used to evaluate the AOA and OS of flaxseed oils. In these studies, the AOA, OS, and content of different BASs are analyzed in order to study the relationship between the indicators and to gain a better understanding of the mechanisms underlying these processes. The study [69] found that the antioxidant activity and ability to absorb radicals deteriorate when oil is heated at higher temperatures. Differences in the antioxidant capacity of flaxseed oil and its fractions were identified in the study [64]. The authors demonstrated that the concentrations of phenolic acids and the antioxidant capacity of superoleic and oleic fractions were higher than those of native flaxseed oil. Additionally, studies [56,65] have shown the presence of various BASs in flaxseed oil, in particular tocopherols, β-carotene, chlorophylls, phenols, and flavonoids, which exhibit antioxidant activity. The role of phenolic acids and antioxidant activity is also demonstrated in the study [72], which established a tendency towards a decrease in oxidative stability in oil obtained from germinated seeds, while the highest concentration of phenolic compounds and antioxidant activity was demonstrated by flaxseed oil from non-germinated seeds. Contrary results are presented in the study [55], in which the authors showed that the germination of flaxseeds increases antioxidant activity and the content of tocopherol, chlorophyll, and carotenoids. The supposed relationship between α-tocopherols contained in oil and its antioxidant activity is shown in the study [58]. The influence of extraction methods on the antioxidant activity of oil from hulls has been revealed. In particular, it was found that oil extracted by supercritical CO₂ showed the highest antioxidant activity, which indicates the ability of oil polarity to influence the oil’s antioxidant activity.

Often, to obtain complete results, methods for determining antioxidant activity are combined with methods for studying oxidative stability. Thus, the authors of [66], evaluating the relationship between antiradical activity and oxidative stability of oils, suggested that DPPH analysis allows predicting the formation of primary (PV, CDs) and secondary (p-AnV) oxidation products in cold-pressed oil. The study [71] showed that the effectiveness of tocopherols in cold-pressed oils was accompanied by a negative correlation between their antioxidant capacity and an increase in PV. A positive correlation was shown between the content of phytosterols and the antiradical ability in the lipophilic fraction of cold-pressed oils. According to the authors, the achievement of high stability is primarily due to the high content of phytosterols. The similarities and differences between the content of BASs (the amount of tocopherols, sterols, and polyphenols), the total antioxidant potential, and oxidative stability were also reported in the study [68].

Studies [57,59,60] have shown the effect of various extraction methods, including the use of additional pressing stages, on the content of BASs, as well as changes in the oxidative indices of the oils obtained. Thus, the use of Soxhlet extraction increased the total oil yield but led to an increase in AV and PV. It was found that the oil obtained in the second pressing stage was of high quality, rich in natural phenolic antioxidants, and could have additional health benefits.

Using the Rancimat (OSI) instrumental method in the paper [55], the authors predicted a more than 4-fold increase in the shelf life of oil from germinated flaxseeds. The authors of [70] found that, depending on the assessment method, the chemical composition of oil can affect its stability. It was determined that all commercially available flaxseed oils used in the study, and purchased within their shelf life, were of good quality.

Compared to other classic OS analysis methods, solid-phase microextraction followed by gas chromatography–mass spectrometry to obtain a profile of volatile compounds formed during oxidation is used much less frequently [55,59]. However, the profiles of volatile secondary products of lipid oxidation obtained can help assess OS mechanisms. For example, a recent study [55] showed that certain compounds, such as heptanal, were found only in conventional oils and were absent in FOCG, which may be related to the conversion of certain lipid oxidation products. At the same time, the profile of volatile compounds (VOCs) might be directly related to the sensory indicators [59] of oils, which allows identifying consumer preferences. At the same time, sensory analysis (SA) can act as an independent method for assessing the OS of oil during storage [64], but it has significant drawbacks and limitations.

According to the conducted literature analysis, the method of Fourier transform infrared spectroscopy (FTIR), despite its prevalence, is rarely used to study the OS of flaxseed oil. The authors of [62] propose the use of a mesh-cell-based FTIR method, which provides a simple, practical, and rapid way to assess the oxidative stability of edible oils when stored under ambient conditions.

Among all the methods and approaches to the analysis of AOA and OS of flaxseed oil that were analyzed, there was a specific mention of the use of electron paramagnetic resonance (ESR) spectrometry. This method can be used to assess both AOA and OS. The study [73] showed the possibility of registering the formation of free radicals in flaxseed oil at different temperatures by evaluating the adducts obtained as a result of the reaction between the free radicals formed and α-Phenyl-tert-butyl nitrone (PBN). This allowed us to establish that peak levels of free radicals were present in flaxseed oil heated to 120 °C, decreasing over 90 min, and absent at the higher temperature of 180 °C.

4.3. The Addition of Biologically Active Substances as a Factor in Increasing the Oxidative Stability of Flaxseed Oil

The process of lipid oxidation is associated with deterioration in taste, loss of nutrients and BASs, and the formation of potentially toxic compounds, which makes food lipids unsuitable for human consumption [74]. In this regard, approaches are being sought to increase the stability of oils that inhibit lipid oxidation. One such approach that has proven successful in preventing oxidation processes is the addition of BASs. The effectiveness of adding BASs to oil is usually determined by analyzing OS, and sometimes additionally assessing AOA, using various methods.

Table 3. Added biologically active substances and parameters used to evaluate the AOA and OS of flaxseed oil.

Oil Type	Added Components	Parameters		Note	Application	Reference
		AOA	OS
FOCD	Polar fractions from flaxseed oil	-	OSI	The addition of a polar fraction containing mainly cyclolinopeptide A led to ↑OSI.	Increasing the OS of FO. Studying the role of CLs in the mechanism of AOA.	[19]
FOCU	Butylated hydroxy anisole (BHA), blend (α-tocopherol, ascorbyl palmitate, citric and ascorbic acids, ethoxylated ethylene glycol)	-	PV/OSI/DSC/ TGA	A complex of methods made it possible to establish that a mixture of antioxidant protected flaxseed oil from oxidation more effectively than BHA. Meanwhile, in samples with antioxidants, ↓PV, ↑OSI, and ↑tonset,TGA/DSC were observed relative to pure oil.	DSC and TGA methods can be used to predict the OS of oils and evaluate the effectiveness of antioxidants.	[75]
FOCU	Rosemary and sage oleoresin, citric acid	-	PV/p-AnV/ CDs/CTs	The use of oleoresins extracted from rosemary and sage and citric acid leads to ↓PV, ↓p-AnV, ↓CDs, and ↓CTs.	Increasing the OS of flaxseed oil. The described model can be used to predict and optimize the effect of antioxidants on the oil’s properties.	[76]
FOD	Hemp extract, α-tocopherol	-	PV/VOCs	The use of hemp flower extract and α-tocopherol leads to ↓PV and reduces the formation of VOCs (by hexanal).	Slowing down oxidation processes in oils with a high degree of saturation.	[77]
FOC	Extract from spirulina and black elderberry, butylhydroxytoluene (BHT), GA	DPPH•	PV/p-AnV	The addition of natural extracts led to ↓PV, and the addition of BHT and GA led to ↑PV during 28 days of storage relative to the control. The value p-AnV↑ during 28 days.	There are advantages to using a mixture of natural antioxidants compared to synthetic ones when assessing the OS of oil.	[78]
FO	Pomegranate seed and peel powder, β-carotene, quercetin GA, tert-butylhydroquinone (TBHQ)	-	OSI	Addition of quercetin, β-carotene, GA, TBHQ (0.01%), and pomegranate seed and peel powder (over 0.1%) led to ↑OSI.	Development of new oils with increased OS.	[79]
FOCU	Vanillin, Hibiscus sabdariffa L. extract, α-tocopherol, BHT	-	PV/AV/CDs	The ability of additives to inhibit oxidation in flaxseed oil decreases as follows: Hibiscus sabdariffa L. extract, vanillin, BHT, α-tocopherol.	Flaxseed oil can be used as a test system for assessing AOA substances.	[80]
FOC	Rosemary extract	DPPH•	AV/PV/ TBARSs	The addition of rosemary extract did not affect the AV, PV, and TBARS values during storage.	The addition of rosemary extract affects secondary oxidation processes, which can be taken into account when storing oils.	[81]
FOC	CoQ, selenomethionine, cholecalciferol, α-tocopherol acetate, α-tocopherol, zeaxanthin, lutein, β-carotene, ascorbic acid esters	-	PV/OSI/AV/ p-AnV	Lutein, CoQ, β-carotene, zeaxanthin in flaxseed oil exhibited pro-oxidant effect (↑PV, p-AnV↑). Addition of α-tocopherol and α-tocopherol acetate (30–150 mg/100 g), and also vitamin D3 (50–150 µg/100 g), did not significantly change the OS of flaxseed oil. Ascorbic acid esters effectively inhibit lipid oxidation processes.	New functional food products based on flaxseed oil that are stable to oxidation.	[82]
FO	Tocopherol, tea polyphenol palmitate, rosemary extract, tea polyphenol extract, antioxidant of bamboo leaves, phytic acid, ascorbyl palmitate	-	PV/OSI/ TBARSs/ESR	The composition 80 mg/kg tocopherol + 40 mg/kg ascorbyl palmitate + 40 mg/kg phytic acid + 240 mg/kg tea polyphenol palmitate had the best antioxidant activity, which increased the shelf life of flaxseed oil by 3.22 times.	Flaxseed oil with improved OS.	[83]
FOC	Ferulic acid, 4-vinylguaiacol, dihydroferulic acid, vanillic acid	-	PV/AV/OSI/ p-AnV/CDs/CTs	Shelf life predictions at 20 °C showed that all tested phenolic additives can be considered effective antioxidants in FOC. However, their AOA depends on concentration (25–200 mg/100 g of oil) and processing temperature (60–110 °C).	Development of new oils with increased OS.	[84]
FOC	Tannic acid, TBHQ, eugenol, β-carotene, α-tocopherol, ascorbyl palmitate, quercetin, L-ascorbic acid, caffeic acid	DPPH•/ CA	PV/OSI/p-AnV	The antiradical activity among hydrophilic antioxidants decreased in the following order: tannic acid, caffeic acid, ascorbic acid; among hydrophobic antioxidants: α-tocopherol, eugenol, β-carotene; among antioxidants with intermediate polarity: quercetin, ascorbyl palmitate. The addition of all natural antioxidants, except α-tocopherol, led to ↓PV and ↑OSI.	Understanding the mechanism of antioxidant action in the presence of minor oil components.	[85]
FOC	Hexyl and palmitoyl esters of sinapic acid	DPPH•/BCB	CDs/CTs/OSI/PV	Palmitoyl sinapate showed the highest AOA. Encapsulation and addition of antioxidants resulted in ↓PV, CDs, CTs, and ↑OSI.	Stabilization of microencapsulated oil.	[86]
FOC	Mullein flower extract (Verbascum nigrum L.)	DPPH•/ABTS	TOTOX/PV/ AV/p-AnV/OSI	The addition of Mullein flower extract increased the oil’s ability to absorb ABTS and DPPH radicals, while increasing OSI, AV, PV (not significantly), p-AnV, and TOTOX.	Development of new oils with increased oxidative stability.	[87]
FOC	Alkyl esters of sinapic acid	DPPH•/BCB	PV/CDs/CTs/OSI	Palmitoyl sinapate showed the lowest PV, while oil stabilized with hexyl sinapate had higher values. It was found that sinapic acid conjugates inhibit the formation of primary oxidation products and slow down the formation of secondary oxidation products.	Development of new oils with increased oxidative stability.	[88]
FOC	Basil, fennel, oregano, rosemary, chili	DPPH•	PV/SA	The addition of spices and herbs led to ↑ antiradical activity and ↓PV. Flaxseed oil with chili had the highest total score (SA) during storage.	Development of new oils with increased oxidative stability.	[89]
FO	Nanofiber zein/basil seed gum/thyme essential oil and nanofiber zein/basil seed gum/encapsulated thyme essential oil	DPPH•	PV/OSI	The addition of nanofibers to oil resulted in ↓PV and ↑DPPH scavenging activity and OSI.	Increasing the shelf life of flaxseed oil.	[90]
FO	Ethylenediaminetetraacetic acid (EDTA), citric acid, rosmarinic acid, vitamin E	-	FR	Encapsulation significantly improved FO stability, both in terms of induction period and oxidation rate (i.e., the slope of the fluorescence-to-time ratio). The stability of the encapsulated oil was slightly improved by rosmarinic acid, while most antioxidants exhibited a pro-oxidant effect.	Incorporating oil into a powder with a high antioxidant content through encapsulation yields an easily added ingredient for enrichment.	[91]
FOC	BHT, TBHQ, 2,5-di-tert-butyl hydroquinone (DTBHQ), propyl gallate, ascorbyl stearate, ronoxan A, 2,20-methylene-bis-(4-methyl-6-tert-butylphenol) (AO-2246), mixed tocopherols 95, α-tocopherol, δ-tocopherol, ascorbyl palmitate, shredded beans, and soybeans	-	OSI/AV/PV/ p-AnV	The addition of all antioxidants studied, except for α-tocopherol, led to ↑OSI at 100 °C. The addition of ascorbyl palmitate (0.04%) to FOC increased OSI at 100 °C from 4.25 to 14.45 h, and at a concentration of 0.02%, it led to ↓PV and ↓p-AnV at room temperature. The addition of shredded beans and soybeans (0.8%) contributed to a decrease in oxidative indices (↓PV, AV, and p-AnV).	Development of new oils with increased OS.	[92]
FSO	SDG, tea polyphenol, resveratrol, caffeic acid, vitamin E, BHT, BHA, TBHQ	-	PV/VOCs	The tested antioxidants showed effective inhibition of oxidation processes in the following order: TBHQ > resveratrol > SDG > tea polyphenol > BHT > vitamin E > caffeic acid > BHA. The use of a combination of 0.01% SDG, 0.01% tea polyphenol, and 0.02% vitamin C inhibits oxidation processes better than 0.02% TBHQ, which increased the shelf life of FSO from 295 to 761 days.	Development of new oils with increased OS.	[93]
FOC	BHT, TBHQ, mixed tocopherols, DTBHQ, ronoxan A, n-propyl-3,4,5-trihydroxybenzene, α-tocopherol, δ-tocopherol, 6-O-palmitoyl-L-ascorbic acid, 2,2′-methylenebis (4-methyl-6-tert-butylphenol), 6-O-stearoyl-L-ascorbic acid	-	PV/AV/p-AnV/OSI	All antioxidants used, except for α-tocopherol, increased the induction period (↑OSI) at 100 °C. The addition of ascorbyl palmitate (0.02%) to flaxseed oil resulted in ↓PV and ↓p-AnV at room temperature.	Increasing the shelf life of flaxseed oil.	[94]
FOCU	Clove essential oil; ginger, allspice, and black pepper extracts; ascorbyl palmitate	-	PV/[ROOH]/ CDs/TBARSs/VOCs	Samples containing clove essential oil and ascorbyl palmitate showed ↓PV, [ROOH], TBARSs, CDs. Ginger, allspice, and black pepper extracts either had no effect on the intensity of lipid peroxidation or exhibited a pro-oxidant effect. All antioxidants led to ↓VOC levels.	Development of new oils with increased OS.	[95]

ABTS^•+—radical 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate); AV—acid value; AOA—antioxidant activity; BCB—β-carotene bleaching activity; BHA—butylated hydroxy anisole; BHT—butylhydroxytoluene; CA—chelating activity; CD—conjugated diene; CLs—cyclolinopeptides; CoQ—coenzyme Q content; CT—conjugated triene; DPPH^•—radical 1,1-diphenyl-2-picrylhydrazyl; DSC—differential scanning calorimetry; DTBHQ—2,5-di-tert-butyl hydroquinone; EDTA—ethylenediaminetetraacetic acid; ESR—electron paramagnetic resonance spectrometry; FR—fluorometric analysis; FO—flaxseed oil; FOC—cold-pressed oil; FOCD—cold-pressed flaxseed oil subjected to desorption; FOCU—unrefined flaxseed oil; FOD—flaxseed oil subjected to desorption; FSO—cold-pressed oil with preliminary heat treatment; GA—gallic acid; OS—oxidative stability; OSI—oxidative stability index; p-AnV—para-anisidine value; PV—peroxide value; [ROOH]—peroxide concentration; SA—sensory analysis; SDG—secoisolariciresinol diglucoside; TBARS—thiobarbituric acid reactive substance; TBHQ—tert-butyl hydroquinone; TGA—thermogravimetric analysis; TOTOX—total oxidation; VOC—volatile organic compound.

presents examples of the addition of various components that have a specific biological activity and influence the oxidation process of flaxseed oil.

According to the analytical review, it was revealed that many studies focus on the use of known antioxidants to inhibit the oxidative processes occurring in flaxseed oil. As is well known, antioxidants are compounds that can slow down the rate of lipid oxidation, so they are widely used to control lipid oxidation and extend the shelf life of vegetable oils. Their antioxidant mechanisms include the removal of free radicals, metal ion chelating, and singlet oxygen quenching [96].

In general, there are three areas of research into the use of antioxidants to inhibit oxidative processes in flaxseed oil:

(1) The addition of known antioxidants, as well as various plant extracts or powders. Thus, studies [77,79,89] demonstrate the positive effect of plant components on oxidative stability. For example, the study [77] shows the positive effect of adding hemp inflorescences as a source of natural antioxidants in oil and lipid products to slow down their oxidation. And in the study [79], it is shown that pomegranate peel and seeds can improve the oxidative stability of flaxseed oil and reduce the severity of effects caused by undesirable temperatures, which can increase the rate of flaxseed oil oxidation. Also, in the study [89], it was shown that the addition of spices and herbs (basil, fennel, oregano, rosemary, and chili) reduced the PV compared to the control sample. It was determined that herbs and spices can remove free radicals and participate in the inhibition of lipid peroxidation.

Besides using plant powders to inhibit the oxidation of flaxseed oil, natural extracts are applied. For example, the use of natural extracts of spirulina and black elderberry prevented oxidation and increased the functionality of flaxseed oil [78]. In the study [80], the effect of Hibiscus sabdariffa L. extract on inhibiting the formation of conjugated dienes in flaxseed oil was demonstrated. A similar study [87] showed that antioxidant activity is directly related to the concentration of added Mullein flowers (Verbascum nigrum L.) extract, and it was found that greater inhibition and activity were related to higher concentrations. In another study [97], oil extracts from sage and cumin leaves can be recommended as antioxidants that provide effective protection against oxidation and extend the shelf life of flaxseed oil and flaxseed-based products. At the same time, it was shown that some of the extracts studied, such as cloves, thyme, or St. John’s wort, can have a pro-oxidant effect. On the contrary, the authors of [95] demonstrated the effect of adding clove essential oil and extracts of ginger, allspice, and black pepper, as well as ascorbyl palmitate. It was found that only clove essential oil and ascorbyl palmitate possessed antioxidant activity in the studied system. The authors recommend clove essential oil as a phenol-containing antioxidant, as well as ascorbyl palmitate as an antioxidant that works effectively at the interface between the lipid and aqueous phases.

Ascorbic acid and its derivatives have been proven to exhibit antioxidant activity. The role of fat-soluble derivatives of ascorbic acid in the protection of flaxseed oil from oxidation is also demonstrated in the study [92]. The authors of [94] also found that fat-soluble derivatives of ascorbic acid effectively protect flaxseed oil from oxidation and significantly increase its shelf life. The stabilizing effect of ascorbyl palmitate is enhanced with an increase in the content of α-linolenic acid (omega-3) in the oil and a decrease in the oxidative stability of flaxseed oil. Kinetic data on the accumulation of peroxide compounds and secondary oxidation products in flaxseed oil with the addition of ascorbyl palmitate during storage at room temperature and free access to air show that the use of ascorbyl palmitate alone is a reliable means of protecting flaxseed oil from oxidative ageing during storage. The study [85] showed that all natural antioxidants were less effective than TBHQ based on mass concentration.

The effect of ferulic acid, a natural phenolic antioxidant, and its derivatives on oxidative stability is presented in the study [84]. The authors, based on the OSI results, found that the oxidative stability of flaxseed oil, predicted at 20 °C, increased linearly with the concentration of ferulic acid, while its derivatives effectively prolonged the induction period at lower concentrations. The addition of phenolic antioxidants generally showed a protective effect. The exception was vanillic acid, which increased the degradation of most bioactive compounds. It is considered that the addition of properly formulated mixtures of ferulic acid and its derivatives can extend the shelf life of flaxseed oil and provide nutritional benefits.

(2) Adding a mixture of antioxidants. Most individual antioxidants are less effective than synthetic ones, which has led to the search for and study of new antioxidants, both individual and in combination.

Thus, the authors of [75] showed that a mixture of antioxidants (a-tocopherol, ascorbyl palmitate, citric and ascorbic acids, ethoxylated ethylene glycol) was more effective than butylated hydroxy anisole (BHA) in protecting flaxseed oil from oxidation processes. OSI and accelerated storage studies showed that flaxseed oil, with the addition of the tested antioxidants at a certain concentration, was more stable to oxidation than other samples [83]. Thus, flaxseed oil containing a combination of tocopherol, ascorbyl palmitate, phytic acid, and tea polyphenol palmitate was more stable to oxidation than other samples and had a 3.22 times longer shelf life than control flaxseed oil. The authors of [82] showed that fat-soluble ascorbic acid esters and their compositions with natural antioxidants based on beans and soybeans were effective and safe stabilizers for flaxseed oil. It was established that the addition of plant-based compositions based on legumes effectively inhibited oxidative processes in flaxseed oil and significantly prolonged its shelf life.

The use of a multi-antioxidant combination consisting of 0.01% secoisolariciresinol diglucoside, 0.01% tea polyphenol, and 0.02% vitamin C showed that it outperformed 0.02% TBHQ, increased the shelf life of flaxseed oil from 295 to 761 days, and delayed the deterioration of sterols and unsaturated fatty acids [93]. The authors claim that this composition may be an effective natural alternative to artificial antioxidants for extending the shelf life of flaxseed oil.

When studying the effect of thyme essential oil in combination with zein nanofibers and basil seed gum on the stability of flaxseed oil, a significant increase in DPPH scavenging activity and OSI was shown [90].

(3) Addition of components isolated from flaxseed oil. It is known that components present in oils can contribute to increased oxidative stability. Thus, the authors of [19] used polar compounds extracted from flaxseed oil as additives for oil with the purpose of enhancing oxidative stability. It was found that flaxseed oil, when passed through silica gel, significantly reduced oxidative stability, which indicates the role of minor BASs in its composition in protecting the oil from oxidation processes. One of the components present in the polar fraction was a mixture of cyclic peptides, which, according to the authors, was responsible for this effect. The authors state that cyclic peptides play an essential role in the antioxidant system of flaxseed oil. At the same time, they can behave as both antioxidants and pro-oxidants, depending on their concentration and the environment.

Besides adding antioxidants to improve the oxidative stability of flaxseed oil, there is also an approach that involves blending several types of oils [79,98]. Thus, the study [79] showed that oil blending (corn, rapeseed, sesame, bitter almond oils) improves the oxidative stability of flaxseed oil and reduces the dependence of the oxidation rate on temperature. A similar effect was found by the authors of [99], who demonstrated that increasing the proportion of black cumin seed oil increased the OSI, as well as the content of BASs (chlorophylls, carotenoids, and total phenolic compounds). The study [100] revealed that blending oils (adding argan oil) in various ratios allows for a controlled assessment of stability and can lead to a significant increase in their resistance to oxidation.

Another approach to inhibiting the oxidative processes of flaxseed oil is microencapsulation [86]. For example, the inclusion of sinapic acid esters in microencapsulated flaxseed oil slowed down the rate of lipid oxidation and increased antioxidant activity. A similar effect, associated with improving the stability of flaxseed oil through encapsulation, is shown in [91].

The application of some approaches described above allows enriching oil with BASs and increasing the oil’s antioxidant potential. It is essential to consider that the additional components used often must be applied in a strictly selected optimal concentration, as well as under specific oil storage conditions; otherwise, they may act as pro-oxidants. Thus, based on the presented studies, the influence of several BASs present in oil (phenolic compounds, tocopherols, phytosterols, etc.) on the antioxidant (antiradical) activity of flaxseed oil has been established. The role of various methods of obtaining flaxseed oil in the content of BASs and antioxidant activity has been revealed. The negative effect of heat treatment on antioxidant activity, the ability to scavenge radicals, and the content of BASs has been shown.

5. Study of the Antioxidant Activity and Oxidative Stability of Flaxseed Processing Products: Flaxseed Meal, Cake, and Hull

In addition to research on whole flaxseed and oil, processed products such as flaxseed meal, cake, and hull are also of considerable interest. The production and processing of flaxseed generate a large amount of waste, known as flaxseed by-products, which may be of interest to various industries due to their rich nutritional profile and different BASs. The main products remaining after flaxseed processing are cake and meal [101]. The use of flaxseed hull may also be promising. Given the diversity of the chemical composition of flaxseed by-products, a range of different methods is used to analyze them, aimed at studying AOA, OS, and BASs.

Table 4. Methods for the study of antioxidant activity, oxidative stability, and the content of biologically active substances in flaxseed meal.

Sample	Methods						Reference
	AOA	Value	OS	Value	BAS	Value
DFM (Extracts)	ORAC DPPH•	0.36–1.07 mmol TE/g 0.081–0.125 g/L	-	-	TPC	7.7–106.5 mg/g	[24]
DFM	ORAC	100.82–136.05	-	-	-	-	[102]
FM (Extracts)	DPPH• ORAC	35.6–63.5% 0.23–0.65 mmol TE/g	PV Aldehyde content	1.6–2.3 mEq/kg 60–190 mmol/L	TPC TTC α-linolenic acid	68.2–92.3 mg GA/g DW 98.2–100% 0.24–57.0%	[103]
FM (Extracts)	RP DPPH• BCB	4240 μg/g 55.28% 73.52%	-	-	-	-	[104]
DFM (Extracts)	TAC RP DPPH• Scavenging of hydrogen peroxide Nitric oxide-scavenging activity	54.44 ± 0.02% 0.0–0.17 30.16 ± 0.80% 25.52 ± 0.075% 24.41 ± 0.39%	-	-	TPC	225 ± 0.025 µg/mg	[105]
FM (Extracts)	ABTS•+ DPPH•	2.95–3.10 mg/mL 3.45–3.95 mg/mL	-	-	TPC * TFC	2.66–2.80 mg GA/g DW 1.20–1.32 mg GA/g DW	[106]
FM (Extracts)	-	-	-	-	TFC * SDG * TPC *	390–1130 μg/g DW 1615.27–3416.95 μg/g DW 4.96–9.85 mg GA/g	[107]
FM (Extracts)	DPPH• ABTS•+ FRAP CUPRAC PCL	9.25 ± 0.68 mg TE/g 12.13 ± 0.61 mg TE/g 61.54 ± 4.98 mg TE/g 75.50 ± 8.02 mg TE/g 191.27 mg TE/g	-	-	TPC TFC	3.80 ± 0.28 mg GA/g n/d	[108]
FM (Extracts)	-	-	-	-	TPC*	14.38–1191.21 mg/kg	[109]
FM and Flaxseed Cake Flour	FRAP DPPH•	59.00–62.00 µmol TE/g DW 11.65–12.28 µmol TE/g DW	-	-	TPC * TFC *	2.64–3.50 mg GA/g 9.06–10.54 mg QE/g	[110]

* The table presents the total content of the determined compound classes. Additionally, the authors of the studies also analyzed the profile of the corresponding compounds. ABTS^•+—radical 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate); AOA—antioxidant activity; BAS—biologically active substance; BCB—β-carotene bleaching activity; CE—catechin; CUPRAC—cupric ion (Cu²⁺) reducing power assay; DFM—defatted flaxseed meal; DPPH^•—radical 1,1-diphenyl-2-picrylhydrazyl; DW—dry weight; FM—flaxseed meal; FRAP—ferric reducing antioxidant power; GA—gallic acid; ORAC—oxygen radical absorbance capacity; OS—oxidative stability; PCL—photochemiluminescence assay; QE—quercetin; RP—reducing antioxidant power; SDG—secoisolariciresinol diglucoside; TAC—total antioxidant capacity; TE—trolox; TFC—total flavonoid content; TTC—total tocopherol content; TPC—total phenolic content.

presents methods for the study of AOA, OS, and BASs in flaxseed meal.

It has been established that in the investigation of flaxseed meal, research is typically conducted by analyzing its extracts in two main directions: investigating antioxidant activity and determining the content of biologically active substances. When analyzing the AOA of flaxseed meal, the primary methods used are DPPH [24,103,104,105,106,108,110], ORAC [24,102,103], ABTS [106,108], and FRAP [108,110]. There are also some less common methods, such as RP [104,105], BCB [104], TAC [105], scavenging of hydrogen peroxide and nitric oxide-scavenging activity [105], PCL, and CUPRAC [108]. When evaluating BASs, TPC [24,103,105,106,107,108,109,110] and TFC [103,106,107,108,110] analysis is often used, while SDG content is evaluated less frequently [107]. It has been found that the content of biologically active substances is generally influenced by the variety of flax [103,106], the type of meal [110], the processing conditions [107], the solvent type [24,103], and the extract fraction used [109]. When analyzing AOA, the following factors influence the result: the solvent type and concentration [24,103,105], the flax variety [102,103,106], and the meal type [110].

Research conducted by the authors of [24] on AOA and BAS content has demonstrated a correlation between ethanol concentration, phenol content, and AOA in the extracts studied. It was found that to obtain flax extract with a high content of phenolic compounds and AOA, the greatest efficiency was achieved when using a mixture of ethanol and water in the proportion of 60:40 (v/v).

When investigating the phytochemical profile of flaxseed meal, Tavarini and co-authors [106] found that flaxseed meal is a good source of SDG lignan and hydroxycinnamic acid glycosides. All extracts demonstrated an excellent radical scavenging activity. However, according to [103], flaxseed meal showed weak antioxidant potential, while refined meal retained its antioxidant activity. It has been shown that AOA was mainly due to a water-soluble system, probably proteins; however, more than one group of flax meal components may be involved in providing the seeds with their efficient and unique antioxidant activity. The study [108] also found strong positive correlations between the antioxidant capacities of fat-soluble compounds. These analyses showed that different types of by-products from processing vegetable oil exhibit high AOA and are rich in phenolic compounds; consequently, their use in bakery products can improve their nutritional qualities.

The presence of phenolic acids in flaxseed meal is shown in the study [109]. It has been established that flaxseed meal is a good source of phenolic acids, both in terms of quality and quantity, and that phenols from all three fractions (i.e., free, esterified, and insoluble-bound) must be taken into account to correctly assess the total phenolic acid content. According to [105], due to its high phenol content, defatted flaxseed meal has demonstrated a wide range of biological activity. The authors of [104] showed that the phenolic extract isolated from flaxseed meal exhibited high antioxidant activity. In another study, the authors of [110] studied and compared the physicochemical, functional, antioxidant activity, and phenolic profile of flaxseed meal and flaxseed cake meal from oilseed cake when roasting flaxseeds in dry air. The use of this roasting type at 180 °C for 10 min increased the free rutin and syringic acid and bound epicatechin and gallic acid, while reducing the caffeic acid, flavonoids, and free resveratrol in the samples studied.

Therefore, roasting improves the phenolic profile and antioxidant activity of the samples studied. The study [102] also showed the effect of seed processing (different roasting temperatures), resulting in a decrease in the antiradical ability of roasted samples compared to untreated ones. A similar study [107] investigated various types of flax processing and identified the optimal extrusion conditions, which resulted in the presence of rutin and an increase in protocatechuic acid content by more than 2 times compared to the control.

It should be noted that, according to the analysis, there is currently an insufficient number of studies on the oxidative stability of flaxseed meal, despite its importance.

Based on the analytical review, it was revealed that studies of AOA, OS, and BAS content in flaxseed hulls are limited and fragmented (

Table 5. Methods for the study of antioxidant activity, oxidative stability, and the content of biologically active substances in flaxseed hull.

Object	Methods						Reference
	AOA	Value	OS	Value	BAS	Value
FH	DPPH•	52.74–78.55%	PV AV p-AnV CDs CTs COX TOTOX	1.28–4.24 meq/kg 1.4–3.2 mgKOH/g 1.23–2.51 1.45–2.64 0.20–0.55 12.49–12.94 4.83–9.88	ChC CaC TPC TFC Vitamin C α-linolenic acid	2.34–65.71 mg/kg 7.52–47.15 mg/kg 62.4–128.3 mg GA/100 g 12.27–17.85 mg luteolin/100 g 1.30–3.20 mg/100 g 48.95–51.52%	[111]
FH (Extracts)	DPPH•	4.95–8.23 g TE/kg	-	-	TPC SDG CouAG FeAG	15.38–32.96 g ferulic acid/kg 16.4–33.9 g/kg 35.7–49.2 g/kg 5.1–15.2 g/kg	[112]
FH (Extracts)	ABTS•+ DPPH• DMPD•+ O2•− scavenging effects FRAP CUPRAC TAC CA	25.67–27.72 μg/mL 49.50–53.30 μg/mL 24.75–28.88 μg/mL 24.75–49.50 μg/mL 0.489–0.525 μg/mL 0.172–0.437 μg/mL 73.95–87.23 μg/mL 8.88–9.24 μg/mL	-	-	TPC p-Hydroxybenzoic acid Vanillin p-Coumaric acid Ascorbic acid Ferulic acid Ellagic acid	3.88–23.30 mg QE/g 120–779 mg/kg 0–8 mg/kg 30–192 mg/kg 9–57 mg/kg 0–71 mg/kg 13–85 mg/kg	[113]

ABTS^•+—radical 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate); AOA—antioxidant activity; AV—acid value; BAS—biologically active substance; CA—chelating activity; CaC—carotenoid content; CD—conjugated diene; ChC—chlorophyll content; CouAG—coumaric acid glucoside; CUPRAC—cupric ion (Cu²⁺) reducing power assay; COX—calculated oxidizability value; CT—conjugated triene; DMPD^•+—N,N-dimethyl-p-phenylenediamine; DPPH^•—radical 1,1-diphenyl-2-picrylhydrazyl; FeAG—ferulic acid glucoside; FH—flaxseed hull; FRAP—ferric reducing antioxidant power; GA—gallic acid; OS—oxidative stability; p-AnV—para-anisidine value; PV—peroxide value; SDG—secoisolariciresinol diglucoside; TAC—total antioxidant capacity; TE—trolox; TFC—total flavonoid content; TOTOX—total oxidation; TPC—total phenolic content.

).

Despite limited research on flaxseed hulls by the authors of [111,112], interesting studies have been conducted showing the influence of the ripening stage and variety of flax from which the hulls were obtained on AOA, OS, and BAS content. For example, in green hulls, the DPPH radical scavenging activity was 52.74%, while in ripe hulls, it was 69.32% [111]. It is worth noting the diverse composition of BASs contained in flaxseed hull (

Table 6. Methods for the study of antioxidant activity, oxidative stability, and the content of biologically active substances in flaxseed cake.

Object	Methods						Reference
	AOA	Value	OS	Value	BAS	Value
FC	ABTS•+ DPPH• FRAP	3.1 ± 0.1 mmol TE/g DW 4.2 ± 0.0 mg GA/g DW 28.3 ± 0.1 μg of ascorbic acid/g DW	CDs PV TBARSs	15.8 ± 0.5 μmol/mg DW 109.2 ± 5.7 μg/kg DW 4.7 ± 0.6 mg MDA/kg DW	TTC * TPC CaC * ChC * α-linolenic acid	2563.0 ± 209.2 μg 100/g DW 484.6 ± 76.1 mg GA 100/g DW 517.0 ± 9.4 μg 100/g DW 137.8 ± 7.5 μg 100/g DW 42.81 ± 0.69%	[114]
FC (Various Extraction Methods)	DPPH• ABTS•+ FRAP RP	29.59–52.96% 52.30–83.10% 0.73–1.09 mmol FeSO4/g 0.27–0.57	AV PV p-AnV CDs CTs	1.66–2.06 mg KOH/g 5.27–95.25 mmol/kg 0.02–90.1 2.33–15.12 0.32–2.13	TPC TFC	32.00–78.01 mg GA/g 1.42–2.73 mg rutin/g DW	[115]
DFC	DPPH• FRAP	7.24–22.54% 1.28–8.67 μmol Fe(II)/g FW	-	-	TFC TPC	5.61–15.64 mg luteolin/100 g FW 475.4–1257.37 mg GA/100 g FW	[116]
DFC (Extracts)	DPPH• FRAP	1.72–11.39% 0.03–1.48 μmol Fe (II)/g FW	-	-	TPC * TFC	108.33–774.33 mg GA/100 g FW 0.08–9.18 mg luteolin/100 g FW	[117]
FC (Extracts)	ABTS•+	10.5%	-	-	-	-	[118]
FC (Extracts)	DPPH•	12.28 µmol TE/g	-	-	TFC * TPC *	10.54 ± 0.18 mg QE/g DW 3.50 ± 0.02 mg GA/g DW	[119]
DFC (Extracts)	DPPH• FRAP	21.15–22.56% 8.50–8.85 µmol of Fe(II)/g FW	-		TPC TFC	1089.68–1128.53 mg GA/100 g FW 11.25–13.80 mg luteolin/100 g FW	[120]
DFC (Extracts)	DPPH•	42.79–76.22%	-	-	TPC	22.84–53.01 mg GA/g	[121]
DFC	-		AV PV FTIR NMR	1.80–3.77 mg KOH/g 0.99–3.51 mEq/kg (depending on storage time)	α-linolenic acid	52.01 ± 0.30%	[122]
DFC (Extracts)	DPPH•	41–73%	-	-	TPC SECO MATA LARI	206–1115 mg GA/L 0.22–7.08 mg SECO/L 0.02–0.06 mg MATA/L 0.02–0.03 mg LARI/L	[123]

* The table shows the total content of the identified classes of compounds, and the authors of the articles also analyzed the profile of the corresponding compounds. ABTS^•+—radical 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate); AV—acid value; AOA—antioxidant activity; BAS—biologically active substance; CaC—carotenoid content; CD—conjugated diene; ChC—chlorophyll content; CT—conjugated triene; DFC—defatted flaxseed cake; DPPH^•—radical 1,1-diphenyl-2-picrylhydrazyl; DW—dry weight; FC—flaxseed cake; FRAP—ferric reducing antioxidant power; FTIR—Fourier transform infrared spectroscopy method; FW—fresh weight; GA—gallic acid; LARI—lariciresinol; MATA—matairesinol; MDA—malonic dialdehyde; NMR—nuclear magnetic resonance; OS—oxidative stability; p-AnV—para-anisidine value; PV—peroxide value; QE—quercetin; RP—reducing antioxidant power; SECO—secoisolariciresinol; TBARS—thiobarbituric acid reactive substance; TE—trolox; TFC—total flavonoid content; TTC—total tocopherol content; TPC—total phenolic content. In studies of flaxseed cake, researchers primarily focus on analyzing its BAS content. In the works [114,118,119], it was shown that flaxseed cake is a valuable source of BASs. At the same time, researchers focus much attention on the conditions of BAS extraction from the cake, such as temperature treatment [115], the use of various solvents [117,123], and ultrasonic exposure [116,121].

). As shown in [112], the contents of SDG, coumaric acid glucoside (CouAG), and ferulic acid glucoside (FeAG) and the profile of phenolic compounds varied significantly depending on the flax variety from which the hulls were obtained. Interesting results were also obtained by a group of authors [113] who investigated the influence of the technology used to produce dry extract (lyophilization or evaporation) from flaxseed hull on the BAS and AOA profiles. It was shown that the BAS profile depends on the method of drying the extract, with the BAS concentration being higher in lyophilized extracts.

Flaxseed cake is another product of flaxseed processing, while presenting a good source of BASs. Table 6 provides examples of BAS content, AOA, and OS assessment methods for flaxseed cake.

The application of various extraction techniques leads to an increase in polyphenol content and, consequently, enhances antioxidant activity. For instance, the study [117] demonstrated that the extraction of phenolic and flavonoid compounds from defatted seed cake depended on the solvents used in the extraction. The mixed solvent system MAW (methanol/acetone/water, 7:7:6, v/v/v) was found to be the most efficient, yielding the highest amount of phenolic and flavonoid compounds in the seed cakes. It was revealed that the antioxidant capacity of the extract was proportional to its phenolic and flavonoid content. A similar pattern was observed in the study [123], which used a 60% ethanol solution as the extraction system.

The use of ultrasonic treatment also leads to an increase in BAS yield and an increase in AOA. The work [116] revealed that ultrasonic treatment at room temperature resulted in a 2-fold increase in polyphenol extraction yield and antioxidant capacity compared to the traditional extraction method. Moreover, the use of heating during ultrasonic extraction resulted in higher polyphenol content in the extracts compared to extraction without heating. Similar results were obtained in the study [121].

The analysis of temperature impact on the yield of BASs from flaxseed cake was evaluated in the study [115]. It was demonstrated that the flaxseed cake extract obtained by hot pressing contained the highest levels of TPC and TFC and exhibited the strongest antioxidant capacity in DPPH, ABTS, FRAP, and total reducing power assays. It was found that various concentrations of the flaxseed cake extract could significantly retard the oxidation of flaxseed oil during storage at 65 °C, with the antioxidant effect being enhanced by increasing the extract dosage.

The changes in AOA and BAS content in the cake during storage are of significant interest [114,122]. The authors of [114] showed that during storage, the content of phenols, carotenoids, chlorophylls, and tocopherols decreased, although α-tocopherol increased significantly due to the possible conversion of γ-tocopherol to the α-form. In addition to assessing the shelf life of BASs, it is worth noting the study of OS in flaxseed cake during storage [122]. The results showed that flaxseed cake is subject to lipid oxidation during storage, but without significant changes in its quality.

Thus, in the study of flax processing by-products such as cake, hull, and meal, a clear research focus is directed towards their AOA and content of BASs. Conducted studies demonstrate that the presented flax processing products are valuable sources of BASs, including phenolic acids, flavonoids, lignans, and other substances with pronounced antioxidant activity. The influence of the solvent on the degree of extraction and the qualitative composition of BASs has been established. It is important to note that among the analytical methods for cake, hull, and meal, assays such as TPC and DPPH are widely employed. These methods allow for screening studies to evaluate the efficiency of the chosen extraction method, followed by a detailed analysis of BAS profiles.

6. Main Methodological Approaches to Studying the Antioxidant Activity and Oxidative Stability of Flaxseed and Its Processed Products

Based on the literature review, the following analysis models can be identified: studies of antioxidant activity: in vitro, in vivo; oxidative stability: in vitro; studies of the AOA of individual biologically active substances contained in flax and flax products: in silico, in vitro, in vivo. This distribution of the use of models is primarily due to their instrumental limitations. The key methods and indicators used to assess AOA and OS in the in vitro model are presented in Figure 2.

According to the literature, the following methods are primarily used to determine AOA in flaxseed and its processed products: a) spectroscopic: ferric reducing antioxidant power (FRAP) assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH^•) radical scavenging, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS^•+), N,N-dimethyl-p-phenylenediamine (DMPD^•+), cupric ion (Cu²⁺) reducing power assay (CUPRAC), oxygen radical absorbance capacity (ORAC); b) chemiluminescence—photochemiluminescence assay (PCL).

To determine OS, the following methods are mainly used: (a) titrimetric methods for measuring peroxide value (PV), acid value (AV), and para-anisidine value (p-AnV); (b) spectrophotometric methods—conjugated dienes (CDs), conjugated trienes (CTs), and thiobarbituric acid reactive substances (TBARSs); (c) instrumental methods for determining the oxidative stability index (OSI)—Rancimat, Oxitest; or (d) chromatographic methods—volatile organic compounds (VOCs). Biologically active substances are mainly determined using spectrophotometric (e.g., to determine total phenolic content (TPC), total flavonoid content (TFC), carotenoid content (CaC), chlorophyll content (ChC)) and chromatographic (e.g., to determine squalene content (SqC), phytosterol content (PSC), vitamin E, total tocopherol content (TTC)).

In addition to the described methods, more specialized approaches are occasionally employed. For oxidative stability studies, differential scanning calorimetry (DSC) [70], electron paramagnetic resonance (ESR) spectrometry [73], FTIR spectroscopy [124,125], peroxide concentration determination ([ROOH]) [95], and calculated indices such as COX (calculated oxidizability value) and TOTOX (total oxidation) may be used [68]. AOA studies can also be performed through β-carotene bleaching assay [126], evaluation of metal chelating capacity [85], electron paramagnetic resonance (ESR) spectrometry [127], and the fluorescence coefficient [91].

Thus, in vitro studies of the AOA and oxidative stability of flaxseeds and their processed products employ a wide range of methodologies. The assessment of oxidative stability in flaxseeds and their products primarily relies on standard determination methods (PV, AV, p-AnV, CDs, CTs, TBARSs, OSI). However, the application of these methods does not fully reflect the processes occurring during oxidation. Their main drawback lies in measuring the total content of primary and secondary compounds with specific functional groups [128]. Consequently, the obtained parameters do not allow for detecting differences in oxidation mechanisms or tracking changes in individual compounds [128]. In this regard, studies utilizing ESR, FTIR, VOC, and NMR methods are being conducted; however, such research on the oxidative stability of flaxseeds and their products remains limited in scope. Future research prospects are associated with expanding the methodological framework, which should enable a more detailed understanding of the ongoing oxidative processes and facilitate the selection of effective inhibition strategies. Furthermore, the oxidative stability of oil- and fat-containing materials is intrinsically linked to antioxidants, both those naturally present in the raw material and those added as supplements.

The main drawback of in vitro approaches for determining AOA is the lack of standardized assessment methods. This absence may be attributed to the specificity of the methods currently in use. Although the DPPH^•, FRAP, and ABTS^•+ assays are commonly used for antioxidant activity assessment, their application is instrumentally limited (e.g., by turbidity formation during the reaction). Furthermore, these assays do not identify the specific compounds responsible for the AOA observed.

In the analysis of the AOA of flaxseeds and their processed products, a preliminary extraction of the antioxidant compounds is typically employed. A significant disadvantage of this stage is the absence of standardized extraction conditions (e.g., the type of solvent or solvent mixture, extraction duration, temperature, and ultrasonic treatment), which leads to variations in the amounts of extracted antioxidants. It is also important to consider that the applied extraction conditions may enhance the extraction of certain antioxidants while causing the degradation of others.

Therefore, to overcome these limitations in AOA determination, it is necessary to develop standardized analytical methods that account for the specific characteristics of the sample under investigation. Additionally, further research into the profiles of antioxidant compounds and the study of individual constituents will help to elucidate their mechanisms of interaction and their respective contributions to the overall antioxidant effects.

Despite the widespread use of in vitro models for evaluating the AOA of flax and its processed products, the results obtained may not fully reflect the effect on the antioxidant status of the living organism. Consequently, there has been active research investigating flax and its derivatives through in vivo studies.

Table 7. Assessment of the AOA of flax and its processed products in an in vivo model.

Sample	Object of Study	Dosage	Experiment Duration	Parameters	Results	Reference
Flaxseed	Wistar albino rats of both sexes	5% and 10% (0.75 and 1.5 g/kg of body weight)	14 days	CAT Peroxidase SOD LPO	Treatment of rats with CCl4 at a dose of 2.0 g/kg of body weight ↓ activity of CAT, SOD, and peroxidase by 35.6%, 47.76%, and 53.0% compared to the control, and the value of LPO ↑ by 1.2 times. The addition of 5% flaxseed to the diet followed by CCl4 treatment caused a recovery of CAT, SOD, and peroxidase by 39.7%, 181.42%, and 123.7%, respectively. The group receiving 10.0% flaxseed showed a recovery of 95.02%, 182.31%, and 136.0% of CAT, SOD, and peroxidase. In the group receiving the toxin without flaxseed, the levels of superoxide dismutase and catalase decreased by 91.4% and 55.33%, respectively.	[129]
Flaxseed with increased content of phenylpropanoid compounds and hydrolyzable tannin	White Giant rabbits	100 g/kg	10 weeks	TAS SOD	The dyslipidemic diet had a negative effect on the lipid profile in rabbits at the 10th week of feeding. Flaxseed of the W86 variety ↑ SOD and TAS activity compared to the group receiving Linola seeds.	[130]
Flaxseed	Female Huoyan geese	5%, 10%, and 15%	56 days	CAT SOD GPx MDA	With an increase in flaxseed concentration, the activity of goose liver enzymes ↑ (CAT, SOD, GPx). MDA content in the goose liver decreased proportionally with higher dietary flaxseed levels. Supplementation of up to 15% flaxseed in the maternal diet resulted in a dose-dependent improvement in the antioxidant status of offspring.	[131]
Flaxseed	Landrace pigs	10%	From 3 to 6 weeks	TAS FRAP dROMs TAC CAT SOD GPx TBARSs	No significant differences in TAS, FRAP, dROMs, or TAC were observed. In the group that received flaxseed for 3 weeks, there was a decrease in SOD, CAT, and GPX activity in the heart compared to the control group. In the group that received flaxseed for 6 weeks, the activity of these enzymes began to increase compared to the 3-week group, but the values were lower than in the control group.	[132]
Flaxseed oil	Male Wistar rats	1 mL/kg of body weight	30 days	TBARSs CAT SOD GPx	The addition of flaxseed oil prevented oxidative damage to lipids and proteins. The oil improved enzymatic antioxidant protection and ↓ glutathione levels.	[133]
Flaxseed oil	Crossbred Hampshire boars (50% Hampshire and 50% Gunghroo)	3% (90 mL)	16 weeks	GPx MDA TAC CAT	The addition of flaxseed oil significantly (p < 0.01) ↑ the concentration of GPx and CAT in blood serum, while the concentration of MDA ↓.	[134]
Flaxseed oil	Wistar rats	1, 2, 3 mL/kg, intraperitoneally	21 days	TBARSs SOD CAT	A dose-dependent inhibitory effect on antioxidant enzymes in heart, liver, and kidney tissues has been shown.	[135]
Flaxseed oil	Teressa goats	25 mL	16 weeks in the rainy season (June to September) and 16 weeks in the dry summer season (December to March)	TAC SOD CAT MDA	It has been shown that adding flaxseed oil to the diet leads to a decrease in oxidative stress levels (MDA↓ and ↑ TAC, CAT, SOD). Meanwhile, high stress levels were observed during the summer season.	[136]
SDG	Male Wistar rats	20 mg/kg of body weight	30 days	TBARSs CAT SOD GPx	SDG prevented lipid oxidative damage and enhanced enzymatic antioxidant defense while increasing total polyphenol content. The study demonstrated that the antioxidant effects attributed to flaxseed are mainly due to its high lignan content, particularly SDG.	[133]
Flaxseed extract	Female BALB/c mice	150 mg/kg, 300 mg/kg, 500 mg/kg	7 days	MDA SOD GPx CAT	The effects of flaxseed extract in the treatment of inflammatory bowel disease (colitis) were studied. Intake of the extract ↓ MDA levels and enhanced antioxidant activity.	[137]
Flaxseed flour	Multiparous lactating Holstein cows fitted with ruminal cannulas	124 g/kg	21 days	TAC MDA TBARSs	The addition of 124 g/kg of flaxseed flour to the diet of dairy cows did not improve the oxidative stability of milk. More research is needed with higher levels of flaxseed flour to assess its potential for improving the oxidative status of cows and preventing milk and plasma lipoperoxidation.	[138]
CLs	Male C57BL/6J mice	10 mg/kg 30 mg/kg	10 weeks	MDA GPx GSH GSSG	CL administration significantly enhanced antioxidant capacity in both liver and pancreatic tissues by ↑ GPx and GSH levels, while reducing GSSG and MDA concentrations.	[139]

CAT—catalase; CLs—cyclolinopeptides; dROMs—reactive oxygen metabolites; FRAP—ferric reducing antioxidant power; GPx—glutathione peroxidase; GSH—glutathione; GSSG—glutathione disulfide; LPO—lipid peroxidation; MDA—malondialdehyde; SDG—secoisolariciresinol diglucoside of flaxseed lignan; SOD—superoxide dismutase; TAC—total antioxidant capacity; TAS—total antioxidant status; TBARS—thiobarbituric acid reactive substance.

presents examples of studies on the AOA of flax and its processed products in vivo studies.

It should be noted that when assessing the antioxidant status of the body in the presented works, the enzymatic components of protection are studied to a greater extent, while the determination of non-enzymatic components is of a more limited nature.

It is known that compounds present in flaxseed and its processing by-products exert antioxidant effects by enhancing the activity of key antioxidant enzymes and reducing the levels of oxidative stress markers. The primary mechanism of the antioxidant action involves the activation of several antioxidant enzymes, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), catalase, and superoxide dismutase (SOD). These enzymes play vital roles in neutralizing reactive oxygen species (ROS) and protecting cells from oxidative damage [140]. A common component of flaxseed capable of scavenging free radicals and reducing oxidative stress at the cellular level is alpha-linolenic acid. Beyond enzyme regulation, flaxseed compounds also provide cytoprotective effects by modulating the activity of key antioxidant proteins, such as nuclear factor erythroid 2-related factor 2 (Nrf2), its inhibitor Keap1, heme oxygenase 1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1) [140]. The activation of these antioxidant pathways contributes to a reduced risk of diseases associated with oxidative stress, including cardiovascular diseases, neurodegenerative disorders, and various forms of cancer [140]. However, a significant limitation of in vivo studies is their labor-intensive nature and the challenge of extrapolating dose–effect relationships to humans. In silico approaches can serve as a preliminary assessment and an additional tool for investigating these mechanisms of action.

Currently, various bioinformatics tools are used to study the physicochemical and therapeutic properties and assess the toxicity, allergenicity, solubility, etc., of individual substances isolated from plant raw materials, including flax. For example, in silico approaches help to obtain information about peptide conformations and mechanisms of interaction between molecules in peptides and improve the properties of peptides [141]. In the study [142], flaxseed proteins and antioxidative peptides were evaluated using a variety of bioinformatic tools in an in silico model. For example, the release of antioxidative peptides was predicted using the BIOPEP software (“search for active fragments”). Most antioxidative peptides showed structural features important for antioxidative activity. Langyan et al. [141] performed screening of new prospective bioactive peptides from flaxseed protein using various online tools: ProtParam from ExPASy (https://web.expasy.org/protparam/) allowed them to evaluate physicochemical properties such as molecular weight, theoretical isoelectric point, total number of negatively charged (Asp + Glu) and positively charged (Arg + Lys) residues, amino acid and atomic composition, extinction coefficient, calculated half-life, total average hydrophobicity, etc.; PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/) was used to calculate the theoretical bioactivity of peptides, the SwissADME tool (http://www.swissadme.ch/index.php#) was used to assess similarity to drugs, and toxicity and allergenicity were assessed using ToxinPred (http://crdd.osdd.net/raghava/toxinpred/) and AllergenFP (http://ddg-pharmfac.net/AllergenFP/), respectively. The authors of [143] constructed the structure of flaxseed proteins using HDOCK software. They identified the mechanism of interaction between flax protein and aldehydes at the molecular level, which lays the theoretical foundation for controlling the taste profile of flax products. Based on the research conducted in [144], CLA and CLE peptides were proposed as the main anti-inflammatory active cyclopeptides that inhibit the TLR4/NF-κB/MAPK signaling pathways, suggesting the potential use of dietary lipids as natural anti-inflammatory supplements. Molecular modeling of flax cyclic peptides using AutoDock Vina has established that inhibition of dipeptidyl peptidase 4 may be a viable pharmacological target for these cyclopeptides. Molecules with anti-inflammatory properties have also been found [145]. In [146], the authors developed a unique scheme for the regulation of mediated microRNA in the biosynthesis pathways of flaxseed components—lignans and cyanogenic glycosides—based on in silico studies. This provides an opportunity to deepen our understanding of the mechanisms of microRNA regulation in these biosynthesis pathways, as well as other specialized metabolites.

As a promising application of in silico modeling for analyzing antioxidant activity, it would be interesting to study the ability of flax-derived compounds to neutralize 2,2-diphenyl-1-picrylhydrazyl radicals using molecular descriptors and a multilayer-perceptron-based neural network approach.

Thus, the use of various bioinformatics approaches contributes to expanding our understanding of the structures, interactions, and potential effects of the objects under study, but the knowledge gained requires experimental verification.

7. Prospects for Research on Flax-Derived Compounds with Antioxidant Activity

Based on the conducted analytical review, it was shown that flaxseeds and their processing products contain a large number of substances with antioxidant activity (Table 1, Table 2, Table 4, Table 5 and Table 6). Figure 3 and

Table 8. The main minor components of flax and its processed products exhibiting antioxidant activity.

Parameter	Seeds	Oil	Meal/Cake/Hull	Reference
TPC, mg GA/100 g	0.6–3315.0	1.5–2120.0	2.6–128.3	[21,23,24,30,31,36,37,41,55,111,114]
TFC, mg luteolin/100g	0.4–689.2 2	11.8–18.8	0.1–17.9	[25,31,41,65,69,75,111,116,117,120]
TSC, mg/kg	n/d	3350.0–5171.7	n/d	[56,68,71]
ChC, mg/kg	n/d	0.6–16.4	2.3–65.7	[55,65,111,114]
α-linolenic acid, %	39.2–58.2	44.9–80.7	49.0–54.9	[23,41,42,82,111,114,147,148,149]
CLs, mg/kg	188.6–623.8	229.3–631.4	385.6–1268.9	[4,150,151,152,153]
SDG, mg/g	0.15–333.0	20.2–51.7	1.6–3.4	[24,28,39,40,58,106,107,112]
SECO, mg/g	0.8–21.7	n/d	0.2–7.8 1	[23,39,40,123,154]
CaC, mg/kg	0.1–6.9	1.6–623.0	5.2–47.2	[33,84,111,149,155,156]
TTC, mg/kg	70.7–747.0	537.0–1065.2	25.6–48.5	[42,72,82,84,147,148,156,157]

CaC—carotenoid content; ChC—chlorophyll content; CLs—cyclolinopeptides; GA—gallic acid; SDG—secoisolariciresinol diglucoside; SECO—secoisolariciresinol; TFC—total flavonoid content; TTC—total tocopherol content; TPC—total phenolic content; TSC—total sterol content. ¹ mg SECO/L; ² mgQE/100 g; n/d—not found.

show the main representative compounds of the antioxidant groups found in flaxseed.

Analysis of the literature revealed that flaxseed and its processing products are a valuable source of a wide range of components exhibiting antioxidant activity. As can be seen from Table 8, their quantitative distribution varied significantly depending on the product type. For instance, in seeds, the TTC values ranged from 70.7 to 747.0 mg/kg. Furthermore, oil was found to be a rich source of these lipophilic antioxidants (537.0–1065.2 mg/kg), whereas in by-products (meal/cake/hull), the values generally did not exceed 48.5 mg/kg.

The carotenoid content differed substantially among the analyzed samples. In seeds, CaC values ranged from 0.1 to 6.9 mg/kg, and in oil, from 1.6 to 623.0 mg/kg. Notably, the seed germination process was accompanied by a significant increase in this parameter, rising from an initial value of 1.6 mg/kg and reaching 5.2 mg/kg on the 5th day of germination [147]. CaC was also detected in flaxseed by-products (5.2–47.2 mg/kg). However, it was demonstrated by the authors of [114] that during the storage of flaxseed cake for 6 months, the CaC decreased from 5.2 to 2.3 mg/kg in plastic containers and to 2.6 mg/kg in paper packaging. The TTC content decreased from 25.6 to 19.8 mg/kg in plastic and to 16.9 mg/kg in paper packaging.

It was also found that seeds and oil contain significant amounts of polyphenolic antioxidant compounds—TPC, up to 3315.0 mg GA/100 g and 2120.0 mg GA/100 g, respectively. TPC values in by-products were significantly lower and varied within the range of 2.6–128.3 mg GA/100 g. Seeds were also characterized by a high TFC value (689.2 mg QE/100 g), while for oils and by-products, these values did not exceed 18.8 and 17.9 mg luteolin/100 g, respectively.

The principal flaxseed lignan, secoisolariciresinol diglucoside, was found in the highest concentrations in flaxseeds (up to 333.0 mg/g) and oil (up to 51.7 mg/g); in processing products, its content did not exceed 3.4 mg/g.

It is known that cyclolinopeptides in flax accumulate primarily in oleosomes [153]. Table 8 shows an almost identical content of these compounds in both seeds (188.6–623.8 mg/kg) and oil (229.3–631.4 mg/kg). Nevertheless, when using certain cultivars, a large amount of these valuable compounds (385.6–1268.9 mg/kg) can also be present in by-products.

Analysis of the alpha-linolenic acid content revealed that the range for flaxseed was 39.2–58.2%; for oil, 44.9–80.7%; and for by-products, 49.0–54.9%. The significant differences are likely due to the development of cultivars with both increased and decreased ALA content, depending on the intended purposes.

To date, compounds such as TSC, ChC, and SECO are analyzed less frequently in flaxseed and its processing products compared to the components described above; however, they are also valuable components possessing antioxidant activity. Thus, the highest SECO content was found in seeds (21.7 mg/g), while sterols were detected only in oil (up to 5171.7 mg/kg). The ChC content was predominant in processing products (65.7 mg/kg).

The identified patterns indicate that the processing of flaxseeds leads to a redistribution of antioxidants, and each of the resulting fractions possesses unique application potential.

Polyphenolic compounds, lignans, tocopherols, and carotenoids are well-studied substances found in flax that exhibit AOA. In this section, we will not focus on the properties of these substances, but more detailed information about their AOA can be found in reviews [2,17,158].

Among the substances contained in flax that exhibit AOA, CLs are of great interest. Although the first cyclic peptide was discovered more than 60 years ago [159], isolation and analysis of properties remain an emerging and poorly studied field. Regarding antioxidant activity, CLs can act as both antioxidants and pro-oxidants. Current research on the AOA of CLs is focused on two main areas: the role of CLs in the oxidative stability of oils, including their use as markers of oxidation, and the study of CLs’ AOA in in vitro and in vivo models. However, greater emphasis is currently placed on investigating the role of CLs in the oxidative stability of oils (

Table 9. Investigation of the role of CLs in the inhibition of oxidative processes in vegetable oils.

CLs	Methods	Results	Reference
Polar fraction containing a mixture of CLs (CLA, CLD, CLE, CLF, CLG)	OSI	The polar fraction containing a mixture of CLs improved the oxidative stability of the oil. Dose-dependent and time-dependent AOA of these peptides were identified. It was established that CLA can selectively interact with metals; consequently, it can inhibit the oxidation process by chelating metal ions.	[19]
CLB, CLC, CLK	AV PV p-AnV TBARSs Aldehyde Ketones Molecular docking	CLB inhibited the oxidation of flaxseed oil (containing Cu2+) at the initial stage of accelerated oxidation, whereas CLK accelerated oxidation. The AOA of CLB and its oxidized form are due to their reducing capacity, as well as their ability to bind with metal ions and intermediate products of fatty acid oxidation.	[160]
CLB, CLC, CLE, CLP, CLJ, CLK	PV p-AnV	CLs themselves exhibited a moderate antioxidant effect on PV, but a weaker effect on p-AnV in flaxseed oil. The methionine content of CLs, in particular CLP, showed a high correlation with the accumulation of primary oxidation products (PV) in the tested matrices, while methionine sulfoxide-containing CLs better reflected changes in secondary oxidation products (p-AnV). The observed consistent correlation between CLP and CLE with the oxidation index of the oil sample indicates their potential usefulness as reliable markers for assessing oil oxidation.	[161]
CLO, CLM, CLN, CLL, CLB	OSI PV	The oxidation of the original, non-oxidized CLs occurs earlier and more rapidly than the oxidation of γ-tocopherol and plastochromanol-8. It has been suggested that CLs provide a certain degree of protection for vitamin E-active compounds. It has been demonstrated that CLs are essentially ingredients for retarding the oxidation of flaxseed oil.	[162]
CLA, CLB, CLC, CLD, CLE, CLF, CLG, CLL, CLM, CLO, CLP	OSI PV p-AnV	CLs containing tryptophan (Trp) exhibit distinct oxidative behavior in the presence of γ-tocopherol. γ-Tocopherol inhibits the oxidation of Trp-containing CLs with methionine (Met) residues and facilitates the oxidation and decomposition of Trp-containing CLs with methionine sulfoxide (MetO) residues.	[163]
CLB, CLC, CLK, CLE, CLJ	AV PV p-AnV	Met-containing CLs oxidize more easily than γ-tocopherol and have specific antioxidant activity. A logarithmic correlation has been found between methionine sulfone-containing CLs and oxidation values.	[164]
CLA	AV PV p-AnV Molecular docking Fluorescence quenching	CLA increases the antioxidant stability of refined flaxseed oil and is capable of slowing down its oxidation by chelating metal ions and intermediate oxidation products.	[165]
CLP, CLB, CLL, CLM, CLO, CLD, CLE, CLC, CLF, CLG	PV AV Off-line MS/MS analysis	The number of Trp and Met residues is crucial for the oxidative stability of CLs. L-ascorbyl palmitate is effective in suppressing the oxidation of both Trp-containing and Trp-free CLs.	[166]

AOA—antioxidant activity; AV—acid value; CLs—cyclolinopeptides; HPLC-MS—high-performance liquid chromatography coupled to mass spectrometry; Met—methionine; MetO—methionine sulfoxide; MS/MS—tandem mass spectrometry; OSI—oxidative stability index; p-AnV—para-anisidine value; PV—peroxide value; TBARS—thiobarbituric acid reactive substance; Trp—tryptophan.

).

Summarizing the results of the study of CLs, it can be concluded that they are essential compounds that contribute significantly to the oxidative stability of flaxseed oil. Their key feature lies in their multifunctionality, which is manifested through direct antiradical activity [160], as well as the ability to chelate metal ions [19,160,165], interact with intermediate oxidation products [160,165], and engage in synergistic relationships with other antioxidants, particularly γ-tocopherol [164,166].

Multidirectional antioxidant activity largely depends on the amino acid composition of peptides. Specifically, Trp-containing CLs have been identified as the most effective in protecting against oxidation [127,163,166].

Notably, the antioxidant efficacy of some CLs diminishes upon their oxidation. This is particularly evident for peptide CLB (containing Met), which is oxidized to peptide CLC (containing MetO), and subsequently to peptide CLK (containing Msn) [150,160]. A distinctive characteristic of Msn-containing peptides is their potential use as reliable indicators for monitoring oxidation processes in flaxseed oil [161,164].

Furthermore, it has been reported that some MetO-containing CLs are a primary factor in the development of bitterness in flaxseed oil due to their accumulation. The resulting bitterness that develops during flaxseed oil storage can be mitigated, for instance, by using specific flaxseed varieties with a lower content of these peptides [167].

The acquired knowledge opens new perspectives for the targeted application of CLs as components that inhibit oil oxidation or as oxidation markers in food and lipid-containing systems.

It should also be noted that studies of the AOA of the initial CLs are currently limited and fragmented. For example, in the work [127], a hypothesis is put forward that the amino acid composition of flax CLs could provide some AOA. The studies showed that all the CLs analyzed exhibited dose-dependent activity in terms of radical scavenging. The reactions of CLA, CLB, and CLC peptides with DMPO-OH led to a 24–30% decrease in ESR signal intensity. The reaction of CLs with more stable DPPH^• radicals demonstrated more complex chemistry than simple scavenging. It was found that peptides containing tryptophan (CLG and CLG”) exhibited higher activity than peptides containing Met and MetO (CLB and CLC). Thus, further study of the AOA of flax CLs is a promising and relevant task, and the knowledge gained as a result will fill the existing gap in this area of research.

8. Conclusions

This review encompasses current knowledge in the field of antioxidant activity, oxidative stability, and the content of biologically active substances in flaxseed and its processed products. It can serve as a reference material for selecting optimal analytical methods.

For the assessment of AOA, the DPPH^•, FRAP, and ABTS^•+ assays are most commonly employed, while PV, AV, CDs, p-AnV, and OSI are typically used to evaluate oxidative stability. These indicators provide only general information, and given that some researchers utilize only one or two methods, the data obtained do not allow for a comprehensive evaluation of the contribution of biologically active substances to antioxidant activity and oxidative stability. Among the most extensively studied biologically active substances in flaxseed and its processed products are phenolic compounds, tocopherols, α-linolenic acid, lignans, flavonoids, cyclolinopeptides, carotenoids, sterols, and chlorophyll. The content of biologically active substances varies significantly depending on the type of product. For instance, seeds were characterized by the highest content of TPC, TFC, the lignan SDG, and its metabolite SECO, and also exhibited high levels of TTC, CLs, and α-linolenic acid. Flaxseed oil had the highest content of TTC, CaC, TSC, and α-linolenic acid, along with high levels of TPC, TFC, ChC, SDG, and CLs. Processed flaxseed products were distinguished by the highest content of ChC and CLs, as well as high levels of CaC, TFC, and α-linolenic acid.

This review did not cover the study of antioxidant activity and oxidative stability in more complex systems (emulsions, microcapsules, food matrices) containing flaxseed and its processed products. Nevertheless, this area is highly relevant for the food industry. Research in this direction would enable the development of products with sufficient resistance to oxidation and improved nutritional and antioxidant properties, which is particularly important for ensuring the population’s access to safe food.