Introduction

In recent years, there has been a growing interest in food and nutrition among people of all age groups. For a variety of reasons, an increasing number of individuals are adopting meat-restricted or meat-free diets. A variety of meatless diets consists of plant-based ingredients, including vegetable oils. A substantial body of evidence suggests that the consumption of vegetable oils has beneficial effects on human health1,2. In this regard, fatty acids (FAs) fulfil a wide range of biological, physiological, and structural functions within the human body3,4. It is therefore essential to ensure that the energy consumed as fat is adequately provided. Current recommendations for total dietary fat intake range from 20 to 35%5,6. Studies have shown both beneficial and adverse effects of fatty acids on the prevention and treatment of human diseases7,8. In nature, fatty acids occur as mixtures of saturated (SFAs), monounsaturated (MUFAs), and polyunsaturated (PUFAs) fatty acids. Consequently, it is essential to analyse the composition of these acids in products in order to assess their nutritional and medicinal value, particularly in fatty-acid-rich foods, dietary supplements, and herbal medicines8,9. Furthermore, the study of nutritional indices is of equal importance to the study of fatty acid composition due to the varying contribution of individual subgroups, including the ratio of n-6 (ω−6) to n-3 (ω−3) fatty acids and polyunsaturated (PUFAs) to saturated (SFAs) fatty acids, atherogenicity index (AI), thrombogenicity index (TI), hypocholesterolemic/hypercholesterolemic ratio (HH) and others8,10,11,12.

Oil plants, particularly species with a high proportion of PUFAs, which the human body is unable to synthesise due to the lack of appropriate enzymes13, are of particular importance in this type of diet. These include linoleic (LA), α-linolenic (ALA) and γ-linolenic (GLA) acids, which constitute the essential fatty acid (EFA) pool2. Other PUFAs can be synthesised from EFAs, provided that they are supplied with food in sufficient quantities and that there is no enzymatic defect in the metabolic pathway of the human system9,14. Consequently, edible oils – obtained from seeds, pulp, or fruit of conventional plants – are a crucial part of human nutrition4,15. During the season 2023/24 (October to September), global vegetable oil production and consumption reached 222.42 and 217.42 million metric tons, respectively, including coconut, cottonseed, olive, palm, palm kernel, peanut, rapeseed, soybean, and sunflower seed oil16. Recently, there has been a growing interest in the use of unconventional oils obtained from nuts (walnuts, hazelnuts, almonds, pistachios), oily herbal plants (borage, evening primrose, safflower, black cumin), fruit seeds (cherry, various berries) and vegetables (carrots), both for direct consumption and for the production of cosmetics17,18,19,20.

In the human diet, vegetable fats represent a significant source not only of health-promoting fatty acids but also of antioxidants, including tocopherols, phytosterols, and phospholipids)4,21. The process of cold-pressing oils allows for the preservation of a significant number of bioactive substances, which is of considerable importance in light of the growing demand for foods with high nutritional and health-promoting value observed among consumers22,23. The susceptibility of cold-pressed oils with high levels of unsaturated fatty acids (UFAs) to oxidation and rancidity may result in a significantly reduced consumption period24,25. Consequently, a growing number of studies are concentrating on techniques that stabilise the chemical composition of oils and improve their oxidative stability, thereby extending their shelf life and even increasing their attractiveness11,26,27. One of the methods involves the obtaining of macerates from oils and plants (vegetables, spices, medicinal herbs) with a variety of properties, including those that are health-promoting (antimicrobial, antioxidant, antiviral, digestive aid) or sensory (taste, colour, or aroma)28,29. Aromatic plants are frequently used in macerates30,31, with existing literature demonstrating their beneficial impact on the chemical composition of edible oils, enhancing both antioxidant activity and sensory properties11,21,29. Given the prevalence of studies on olive oil, there is a scarcity of literature examining the impact of the maceration process on the fatty acid composition of alternative edible oils32,33,34. In contrast, the impact of prolonged storage on the fatty acid profile has recently been extensively discussed, even in the context of cold-pressed oils derived from non-conventional plants35,36. Nevertheless, there is a deficiency of studies examining the combined impact of maceration and long-term storage on the fatty acid composition11,37. Accordingly, the present study was designed to examine the fatty acid profile of cold-pressed oils obtained from eight diverse species (black cumin, safflower, borage, evening primrose, hazelnut, walnut, sea buckthorn, and oilseed rape) and to evaluate the influence of maceration and long-term storage on their composition.

The objective of this study was to evaluate the impact of herbal additives, including basil (Ocimum basilicum L.) or sage (Salvia officinalis L.) leaves in different doses, as well as the impact of long-term storage (10-month refrigerated storage), on the fatty acid composition, nutritional indices (AI, TI, HH, PUFAs/SFAs, and ω−6/ω−3) and total low molecular weight antioxidant activity (TAA) of oils cold-pressed from seeds and berries of selected oilseed species important for nutritional and pharmaceutical purposes. The obtained results represent a significant contribution to the current understanding of how maceration and storage affect the fatty acid profiles of unconventional cold-pressed oils. The rationale for employing macerates prepared from fresh leaves originated from suggestions provided by an edible oil producer (“Dolina Iwełki”, Nienaszów, Poland) collaborating with us, who indicated a preference for flavoring with fresh plant material due to the fresher aroma of the final product and their established procedures for controlling microbiological instability. According to the available scientific literature, fresh herbal material, such as basil, contains substantially higher levels of antioxidant compounds compared to dried material. These include linalool, limonene, 1,8-cineole, among others38. In addition to enhancing the oxidative stability of the oil, enrichment with such compounds also improves the sensory properties of the product39.

Results and discussion

Cold-pressed oils represent a valuable source of polyunsaturated fatty acids and other substances with high biological activity40,41. In recent years, the range of edible vegetable oils has been expanding continuously, and currently is no longer limited only to the country of origin. Instead, it is now a global phenomenon17,41,42,43. Additionally, flavoured plant oils (mainly olive oil) are being produced to maintain high quality, improved stability, enhanced sensory characteristics, and beneficial health properties11,44. These oils are typically produced through the infusion or maceration of spices into the oil34. To the best of our knowledge, there is a limited number of studies examining the effects of both maceration and long-term storage on the fatty acid profile and nutritional indices of the selected cold-pressed oils. Consequently, we investigated whether the addition of sage or basil to base oils and long-term refrigerated storage would affect the properties of the oils.

Fatty acid profile of the tested oils and macerates

The composition of fatty acids and the presence of natural antioxidants (including phenolic compounds, tocopherols, ascorbic acid, squalene, rutin, carotenoids, flavonoids, vitamins and anthocyanins) are among the factors that contribute to the high quality and stability of oils45,46. The fatty acid composition of oils can be altered by a number of factors, including cultivation conditions, harvest time, environmental conditions (especially temperature), seed treatment prior to extraction and processing conditions47,48,49,50,51. When analyzing oils, varietal factors should also be considered, as the yield and composition of oils from the same variety grown in the same location can vary from year to year52. Therefore, the content of bioactive compounds, including fatty acids, can also vary slightly in a given oil, as confirmed in our study (Fig. 1, Tab. S1-S3).

Fig. 1
figure 1figure 1

The impact of herbal additives (sage – Salvia officinalis L. or basil – Ocimum basilicum L.) and long-term storage (10 months at 8–10 °C) on the fatty acid (FA) composition in cold-pressed oils derived from the seeds and berries of eight diverse plant species: (a) black cumin seed oil; (b) safflower seed oil; (c) borage seed oil; (d) evening primrose seed oil; (e) hazelnut oil; (f) walnut oil; (g) sea buckthorn-rapeseed oil; (h) rapeseed oil. Control oil refers to cold-pressed base oil without any additives. Selected FAs include: C16:0 – palmitic acid; C18:0 – stearic acid; trans-C18:1n-9 – elaidic acid; cis-C18:1n-9 – oleic acid; cis-C18:2n-6 – linoleic acid; C18:3n-3 – α-linolenic acid; C18:3n-6 – γ-linolenic acid; C22:1n-9 – erucic acid. All analysed FAs are presented in Table S3, including those constituting less than 2%. Analysed FAs were categorized into groups and summed: the total content of saturated (Σ SFAs), polyunsaturated (Σ PUFAs), monounsaturated (Σ MUFAs), and essential (Σ EFAs) FAs. Values marked with the same letters (separately for each fatty acid and species) are not significantly different according to Duncan’s test (P ≤ 0.05; n = 3).

Full size image

The stability of cold-pressed oils is limited by the content of PUFAs, especially α-linolenic acid, as well as the content of antioxidants, and is usually set for 6 or 12 months25. Oils with a high content of SFAs are typically more durable, yet their health-promoting benefits are significantly inferior, as such fats contribute to elevated levels of low-density lipoprotein (LDL) and cholesterol in the blood53. Consequently, an alternative method for enhancing the resistance of cooking oils to oxidation is to enrich them with substances that possess antioxidant properties, which, through self-oxidation, inhibit the oxidation of unsaturated fatty acids. The application of natural antioxidants from spices and herbs may be one method of improving the oxidative stability of cold-pressed oils. These antioxidants deactivate free radicals in the first stage of oxidative changes, thereby preventing or inhibiting the development of further reactions. The addition of sage or basil to our base oils, apart from enhancing the flavour and nutritional qualities of the test oils, was aimed at determining whether their addition affected oil storage stability in terms fatty acid composition. Cluster analysis grouped 80 oils (64 herbal macerates and 16 control oils) into three main clusters mostly based on polyunsaturated linoleic acid and monounsaturated oleic acid content: (1) hazelnut (H), rapeseed (R), and sea buckthorn-rapeseed (SB-R) oils rich in monounsaturated oleic acid; (2) borage (B) seed oil with high levels of polyunsaturated linoleic acid and γ-linolenic acid, and monounsaturated oleic acid and saturated palmitic acid; and (3) walnut (W), evening primrose (EP), black cumin (BC), and safflower (S) oils dominated by polyunsaturated linoleic acid (Fig. S1). The principal component analysis revealed that the BC seed oil showed the greatest changes across both principal components in response to long-term storage and herbal additives (Fig. S2).

In our research, the control oils H, R, S, and W had a typical fatty acid composition recommended by the Codex Alimentarius standard22 and all the control oils tested (H, R, S, W, EP, BC, B and SB) had a similar composition as described by other authors36,41. Our investigation focused on two types of oils: those dominated by PUFAs (the seed oils of BC, S, B, EP, and W) (Fig. 1a, b, c, d, f) and those dominated by MUFAs (H, SB-R, and R oils) (Fig. 1e, g, h). The FA composition of control oils and macerates from different plant species was examined both before and after 10 months of storage (Fig. 1a-h). The investigated macerates and control oils differed slightly in FA composition, observed mainly in BC seed oil and SB-R oil.

In BC seed oil, the dominant FA was linoleic acid (LA; cis-C18:2n-6) (55.2%), followed by oleic acid (OA; cis-C18:1n-9) (21.8%), palmitic acid (PA; C16:0) (13.1%), and stearic acid (SA; C18:0) (3.0%) (Fig. 1a), as confirmed by the results of other authors54,55,56,57,58. The most visible changes in FA profile of BC seed oil were noticed in the percentage content of polyunsaturated LA and saturated PA (Fig. 1a). The addition of 50 g and 100 g of basil caused a slight but significant change in the FA composition of the oil. An increase (approximately 3% points) in the percentage of LA in the macerates with basil was observed in comparison with control oil, regardless of storage conditions and basil dose (Fig. 1a). The opposite observation was made for PA, which decreased in basil macerates by 4–5% points in comparison with control oils, regardless of the dose or storage conditions (Fig. 1a). The sage addition also reduced PA percentage content, although to a much lesser extent than basil. Overall, the effect of sage addition on FA composition was minor in the case of BC seed oil compared with basil addition. The storage effect was minor for all FAs in BC seed oil and macerates (Fig. 1a). The changes in SFAs content corresponded to the changes observed for PA, indicating that the herbal additives reduced SFAs percentage concentration; however, the storage effect was minor (Fig. 1a). While the herbal additives decreased SFAs in macerates, both sage and basil significantly increased the percentage concentration of PUFAs regardless of the dose, but no storage effect was noted (Fig. 1a). MUFAs were only slightly positively affected by the additives and storage conditions compared with PUFAs and SFAs (Fig. 1a). Finally, EFAs content was also higher in macerates compared with control oils, regardless the storage conditions (Fig. 1a). The storage did not affect the EFAs in BC seed oil (Fig. 1a).

In safflower seed oil, polyunsaturated LA (73.7%) was also the dominant fatty acid, followed by monounsaturated OA and saturated PA and SA, which were present in much smaller amounts (11.5%, 7.4% and 3.1%, respectively). Similar results were reported by several authors35,59,60,61,62,63,64. The changes in the FA profile of S seed oil, in terms of herbal additives and storage effects, were minimal (Fig. 1b). Macerates of safflower seed oil with basil (100 g) both before and after storage had a higher percentage of PUFAs than control oils (Fig. 1b).

The dominant FA in B seed oil was LA (31.7%), followed by γ-linolenic acid (GLA; 18:3n-6) (23.4%), OA (17.1%), PA (12%), SA (4.2%) an erucic acid (ERA; C22:1n-9) (2.8%) (Fig. 1c), as supported by a large body of other research17,64,65,66,67,68. Regarding herbal additives before storage, only the 50 g dose of sage significantly increased the LA amount in the FA pool of the macerate compared with the control oil (Fig. 1c). In the case of BO seed oil, the storage effect had a more significant impact on the FA profile than the herbal additives. After 10 months of storage, the control oil showed a significant increase in LA, OA, and PA percentage content, along with a significant decrease in the percentage of GLA, compared with the control oil before storage (Fig. 1c). Overall, the analysis revealed that long-term storage led to a significant increase in the percentage content of LA and OA in the FA pool but a significant decrease in GLA (Fig. 1c). No clear modifications were observed in terms of total contents of SFAs, PUFAs, MUFAs and EFAs (Fig. 1c). In the case of borage seed oil, generally no significant differences were found between macerates and control oil before and after storage (Fig. 1c).

The dominant fatty acid in EP seed oil was LA, comprising 72.4%, while GLA was present at a smaller percentage of 10.8% (Fig. 1d). PA and OA were also found, contributing 6.1% and 5.6% of the FA pool, respectively (Fig. 1d), as confirmed by several research studies56,64,66,67,68. Similarly to B seed oil, the effect of storage had a more significant impact on the fatty acid profile than the herbal additives. After long-term storage in the control oil, as well as in the sage and 100 g basil macerates, a significant increase (1.4–1.8% points) in the percentage of LA was observed (Fig. 1d). In contrast, GLA decreased after storage in the control oil and all macerates (Fig. 1d). Total contents of unsaturated and saturated FAs remained almost unchanged after storage. No significant differences in the percentage of PUFAs were observed in evening primrose seed oil and macerates after long-term storage. The only exception was 10-month stored macerate with basil (50 g), which was characterized by a significantly lower percentage of PUFAs than in the control oil after storage (Fig. 1d).

The dominant fatty acid in H oil was OA (76.9%), while PA and LA were found in much smaller amounts (7% and 6.5%, respectively), which was confirmed by studies by Celenk et al.69 or K. Król et al.36. Small amounts of SA and elaidic acid (ELA, trans-C18:1n-9) were also found (Fig. 1e). The herbal additives caused no significant differences in the FA composition compared with the control oil, regardless of the dose or storage period. However, after 10 months of storage a significant increase in the percentage of the monounsaturated OA was observed in the control oil and in the sage macerate (50 g), with a similar change found in the MUFAs (Fig. 1e). Hazelnut oil macerates with basil (50 g and 100 g), both before and after storage, were characterized by a significantly higher percentage of MUFAs than in the control oil before storage.

In walnut oil, the dominant FA was LA (58.8%), followed by OA (16.4%), α-linolenic acid (ALA, 18:3n-3) (11%), PA (7.2%), and SA (3.0%), similar results have been obtained in other studies17,20,56,64,70. The addition of herbs had no impact on the FA composition of W oil before storage; however, this changed following long-term storage. Namely, after storage in macerates with sage (50 g) and basil (50 g and 100 g), there was a decrease in the percentage of LA and ALA in the FA pool compared with the control oil (Fig. 1f). Consequently, in the basil macerates discussed above, a decrease in the total PUFAs and EFAs by 1.8 to 2.7% points was noted (Fig. 1f). In this context, similar changes were observed for LA, PUFAs and EFAs as a storage effect when comparing the basil macerates before and after storage (Fig. 1f). For other FAs and their groups, both effects were minimal.

The predominant FA in sea-buckthorn-rapeseed oil was OA, representing 66.3% of the total FA content. The remaining FAs, namely LA, PA, ALA, ELA and SA were present in smaller amounts, accounting for 10.2%, 7%, 5.2%, 2.3%, and 2.2% respectively. The addition of sage (100 g) and basil (50 g and 100 g) significantly reduced the level of OA compared with the control oil (Fig. 1g). Before storage, the decrease ranged from 3.3% to 6.4%, while after storage it ranged from 4.8% to 7.4% (Fig. 1g). In both instances, the reduction was more pronounced with the 100 g dose. The LA percentage content, in contrast to OA, increased significantly after adding sage (100 g) and basil (50 g and 100 g) regardless of the storage conditions (Fig. 1g). Before storage, the increase ranged from 3.0% to 5.1%, while after storage it ranged from 2.6% to 4.8% (Fig. 1g). As with OA, the increase was more pronounced at a dose of 100 g in both cases. The addition of sage at a dose of 50 g had no significant effect (Fig. 1g). Consequently, total PUFA and EFA percentage contents increased by 3.4–6.3% points, whereas MUFA contents exhibited a decrease of 3.5–6.8% points (Fig. 1g). Following prolonged storage, the control oil exhibited elevated levels of OA within the FA pool compared with pre-storage conditions (Fig. 1g). In sage and basil macerates, the OA also increased significantly, but by approximately 2–2.5.5% points, whereas in the control oil, it increased by 3.6% points. Therefore, a comparable effect was observed for total MUFAs. The impact of storage was less pronounced than that observed for total PUFAs and EFAs, whose percentage contents decreased by approximately 1.0% points in control oil and by approximately 1.5% points in macerates (Fig. 1g). In comparison with the impact of herbal additives on the LA percentage content, prolonged storage resulted in minimal changes to its content (Fig. 1g). For the FAs PA, SA, ELA, and ALA, and total SFAs, both effects were negligible (Fig. 1g). The SB-R macerates, sage (100 g) and basil (50 g and 100 g), before and following 10-month storage were characterized by lower percentages of MUFAs in comparison with the control oils. In the case of sage macerate (50 g), total MUFAs level was significantly higher than in the control oil before storage but lower than in control oil after storage (Fig. 1g).

The most prevalent fatty acid in R oil was OA (60.3%), followed by LA (16.4%), and ALA (6.6%), PA (5.1%), and ELA (3.4%) (Fig. 1h), which support the findings of Chew71 and Symoniuk et al.72. The addition of herbs had no impact on the composition of FAs, in contrast to the effects of long-term storage, which resulted in significant differences to the FA profile (Fig. 1h). Notably, a significant increase of 3.4–4.1% in the percentage content of OA in the FA pool was observed in both the control oil and macerates after 10 months of storage (Fig. 1h). In contrast, the percentage content of ELA in the control oil and macerates decreased by 1.5–1.9% after storage (Fig. 1h). As a consequence, the total MUFAs increased by 1.5% in control oil and by 1.8–2.1% in macerates (Fig. 1h). For other FAs like PA, LA, and ALA, and total sum of SFAs, PUFAs and EFAs, both effects were minimal (Fig. 1h). In the case of oilseed rape oil and macerates, 10-month storage resulted in a significant increase in the percentage of MUFAs in comparison with the control oil and macerates before storage (Fig. 1h).

The observed changes in the proportion of PUFAs, especially linoleic acid, after basil and sage supplementation are consistent with previous findings showing that phenolic compounds and essential oils from Lamiaceae herbs (e.g., Ocimum basilicum L. and Salvia officinalis L.) exhibit strong antioxidant activity73,74,75. It can be hypothesised that these compounds may contribute to the maintenance of more favourable fatty acid proportions following slow screw pressing of the oil. The method of oil processing significantly impacts the oxidative stability of the final product25. Due to the low level of processing involved in producing them, cold-pressed oils contain more pro-oxidant components than refined oils, including metal ions, chlorophylls and lipid peroxides23. Cold-pressed oils frequently exhibit an elevated initial autooxidation state and less predictable oxidative stability23. Antioxidant mechanisms, such as free radical scavenging, are known to reduce lipid peroxidation processes73,74. Such mechanisms would be highly desirable in cold-press oils, which are often rich in PUFAs, such as black cumin seed oil. The increased proportion of PUFAs may explain the decrease in the proportion of SFAs, which are considered relatively stable fatty acids76. It also might by hypothesised that, as with black cumin oil, the low processing level of cold-pressed sea-buckthorn–rapeseed oils increase their susceptibility to oxidation due to residual pro-oxidants23, highlighting the importance of exogenous antioxidants in shaping fatty acid stability. Highly PUFA-rich oils like safflower, borage, evening primrose undergo rapid peroxidation that may exceed the protective capacity of the added compounds, resulting in no measurable change in relative fatty acid proportions.

The FA profile of control oils derived from various plant species and their macerates was analysed both before and after 10 months of storage, revealing differences in FA composition. The studies conducted by Ambrosewicz-Walacik and Tańska77 on extra virgin olive oil (EVOO) demonstrated that the storage of laboratory-flavoured EVOO, with estragon, ginger, colour peppercorns or thyme, for 60 days at 8–20 °C did not result in any significant change in the concentration of the principal fatty acids, namely OA and LA. In contrast, the authors observed the opposite effect in commercially flavoured EVOO, with lemon aroma, basil or ginger and chilli, where OA was lower and LA was higher after storage than in EVOO without additives. Moreover, this effect was also present prior to storage. The outcomes on commercially flavoured EVOO were analogous to those observed in our studies on SB-R oils specifically, the comparison of OA and LA levels between the macerates and the control oil. In a further study on EVOO, Benkhoud et al.11 investigated the impact of incorporating six essential oils. An objective of the study was to ascertain the effect of these additions on the fatty acid composition and nutritional value of the EVOO. The findings indicated that the nutritional value of the EVOO was effectively preserved by the additives. Following a 12-month storage period at 20 °C, the flavoured EVOOs, which had been infused with rosemary, thyme, fennel, Brazilian black pepper, black pepper or orange flavours, exhibited a higher LA content and a lower OA content in comparison with the control oil. These findings are in accordance with the results obtained in our study for SB-R oil. Additionally, Sacchi33 investigated the effect of flavouring EVOO on its fatty acid profile, yet without considering the effect of storage. The impact of fresh lemon addition was found to be minimal, which aligns with other outcomes observed for EVOOs32,34, and the FA profile in macerates of borage and rapeseed oil in our study.

In contrast to the results observed for EVOO, herbs did not demonstrate a preventive function with respect to the EFAs of certain oils in our study, as evidenced by a decrease in the GLA content. GLA, which belongs to the n-6 group, occurs in evening primrose and borage oils40,68,78,79, as confirmed by our research. Both species are classified as medicinal plants and their oils are applied in the prevention and treatment of numerous diseases65. Additionally, the high concentration of GLA may contribute to the reduction of cholesterol levels80. The findings of our studies indicate that the GLA content in both EP and B was influenced by the storage duration, resulting in a notable decline in GLA levels (Fig. 1c, d). However, the addition of herbs had no significant impact on this result.

Nutritional indices

The role of fatty acids in human and animal health is of paramount importance in determining the appropriate type of fat to consume. Fatty acids can be obtained from a variety of dietary sources, each with unique properties. It is widely acknowledged that variations in the fatty acid composition of oils significantly influence the functionality and characteristics of oils. Therefore, a careful analysis of the fatty acid composition of these sources is essential. Plaha et al.12 highlighted that nutritional indices such as the ω−6/ω−3 PUFA ratio, MUFA/PUFA ratio, PUFA/SFA ratio, desirable fatty acids, along with lipid health indices including the atherogenic index (AI), thrombogenic index (TI), oxidability index (COX), oxidative stability (OS), hypocholesterolemic/hypercholesterolemic index (H/H), and peroxidability index (PI) are valuable tools for assessing the nutritional and medical significance of various foods and raw materials. The importance of these nutritional indicators for various products, including plant oils, has been extensively discussed in numerous scientific articles1,8,81,82,83. In order to assess whether the nutritional value of the tested oils and macerates changed after storage, the following parameters were considered: the ratio of ω−6 (C18:3n-6, C18:2n-6,9, C18:2n-6) and ω−3 (C18: 3n-3) fatty acids (ω−6/ω−3), the atherogenicity index (AI), the thrombogenicity index (TI), the hypocholesterolemic/hypercholesterolemic ratio (HH) and the ratio of total polyunsaturated to saturated fatty acids (PUFA/SFA) (Tab. S4, S5).

The heatmap and clustering analysis (Fig. 2) revealed three primary clusters, largely attributed to variations in the fatty acid profiles. The first group consisted of W, R, SB-R, R and H oils (Fig. 2), which shared a common feature of low ω−6/ω−3 ratio and low AI and TI indices. However, R oil displayed the highest HH ratio among all oils tested, while W oil exhibited the highest PUFAs/SFAs ratio within this group (Fig. 2). Minor differences were observed in the effects of herbal additives and long-term storage. The most notable differences were found in the R and SB-R oils. In R oils, higher HH values were noted after long-term storage, regardless of herbal additives. In contrast, for SB-R oils, the basil macerates, particularly those with a 100 g basil addition, exhibited the lowest HH value both before and after storage (Fig. 2). The second group of oils comprised S and EP seed oils, as well as all basil macerates of BC seed oil (Fig. 2), which demonstrated the greatest diversity in terms of the nutritional indices. These oils exhibited low or average values for AI and TI. However, EP oils showed a high HH index and high ratios of PUFAs/SFAs and ω−6/ω−3. In contrast, S oils exhibited low HH values and average or high ratios of PUFAs/SFAs and ω−6/ω−3. In basil macerates of BC seed oil, regardless of storage conditions, the HH index was found to be low, as were the ratios of PUFAs/SFAs and ω−6/ω−3. The final group comprised B seed oils, BC control oils and BC sage macerates, which exhibited the highest AI and TI values, low values of the PUFAs/SFAs ratio, and the lowest HH values. The sage macerates of BC seed oil (both before and after storage, regardless of dose) and the basil and sage macerates of B seed oil before storage demonstrated low ω−6/ω−3 ratios. In contrast, average values were observed in the BC control oils and in storage-treated herbal macerates of B seed oil.

Fig. 2
figure 2

Heatmap and clustering analysis of nutritional indices in cold-pressed base oils derived from the seeds and berries of eight diverse species and their herbal macerates, stored for a prolonged period (10 months at 8–10 °C). Analysed oils: black cumin (BC) seed oil, safflower (S) seed oil, borage (B) seed oil, evening primrose (EP) seed oil, hazelnut (H) oil, walnut (W) oil, sea buckthorn-rapeseed (SB-R) oil, and rapeseed (R) oil. Control (base) oil—cold-pressed oil without any additives. Macerate – oil obtained from the maceration of 50–100 g of leaves from either sage (Salvia officinalis L.) or basil (Ocimum basilicum L.). Analysed nutritional indices: ω−6/ω−3 – the ratio of ω−6 (C18:3n-6, C18:2n-6,9, C18:2n-6) and ω−3 (C18:3n-3) fatty acids; AI – atherogenicity index; TI – thrombogenicity index; HH – Hypocholesterolemic/hypercholesterolemic ratio; PUFAs/SFAs – the ratio of total percentage content of polyunsaturated and saturated fatty acids. The colour scale in heatmap (b) illustrates the Z-score. Mean values and F-statistics are presented in Table S4 and Table S5, respectively.

Full size image

The rise of agribusiness and modern agriculture has led to a significant increase in ω−6 PUFA intake in the diet, while simultaneously reducing ω−3 PUFA levels84. According to the nutritional recommendations, the optimal ratio of n-6 (ω−6) to n-3 (ω−3) acids in a healthy diet that positively affects inflammation and other biological processes should be in the range of 3–5 to 1, while these proportions in the diets of many European and other countries are skewed, with the percentage of ω−6 acids being greatly exceeded7. Fatty acids from the n-3 and n-6 families belong to the group of EFAs that must be supplied to the body with food13. Fatty acids from the n-3 group prevent inflammation, can be used in the treatment of certain neoplastic diseases, are necessary for the proper functioning of the body, especially the nervous system, but their proportion in the daily diet is still too low10,85,86. FAs from the n-6 group are important hormone source for humans which, apart from their beneficial effects, also show negative influence. Their excess inhibits EPA acid absorption and stimulates inflammation, and promotes the occurrence of cardiovascular diseases, autoimmune diseases, obesity, and cancer growth8,82,84,87. The most favourable ω−6/ω−3 fatty acid ratios (ranging from 1.95 to 5.72) were found in the case of R, SB-R and W oils. In contrast, the remaining oils exhibited notable deviations from the optimal values, with the ω−6/ω−3 ratio spanning a range from 30.42 for H oil to 661.88 (control)/689.29 (after storage) for EP oil (Tab. S4).

As stated by Kostik et al.88, the PUFA/SFA ratio is a significant factor in determining the nutritional value of oils. A ratio value exceeding 1 is indicative of a product’s nutritional value. A number of studies have demonstrated that a higher PUFA/SFA ratio is associated with a reduction in lipid deposition in the body8. The PUFA/SFA index values for the oils and fats under examination are presented in Tab. S4. The highest value for the PUFA/SFA index was observed for EP oils and their macerates (8.67–9.67), while the lowest value was noted for H oils and their macerates (0.56–0.68) (Fig. 2). In light of the limitations of the PUFA/SFA ratio in evaluating the atherogenicity of foods, Ulbricht and Southgate89 proposed a new index, IA, derived from the PUFA/SFA ratio8. As previously proposed by Ratusz et al.90 and Ying et al.19, the nutritional indices employed in the present study (AI, TI and HH) are more effective than fatty acid composition alone in evaluating the nutritional value of oils. These nutritional indices provide insights into the differential effects of individual fatty acids on human health8,82. The AI and TI are widely recognized as reliable indicators for assessing the potential cardiovascular effects of FAs in fats and oils8,90, particularly regarding atherosclerosis risk and thrombus formation89. Previous research81 has demonstrated that lower AI and TI values correlate with reduced coronary heart disease risk. Therefore, lower values indicate greater health benefits, suggesting potential for cold-pressed oils1. The HH ratio serves as an indicator of cholesterol metabolism. Literature data suggest that oils with higher HH indices are more favourable for human dietary consumption8,90. In the present study, both the cold-pressed oils and their macerates exhibited consistently low AI (0.06–0.18) and TI (0.11–0.4) values (Tab. S4), suggesting potential cardioprotective properties of their constituent fatty acids. Among the analysed samples, R and EP seed oils and their macerates demonstrated the highest HH indices (15.39–16.82 and 12.5–13.82, respectively), while B and BC oils and their macerates showed comparatively lower values (Tab. S4). The most significant impact of herb addition on the nutritional indices of the oils under examination was observed in the case of BC oil. The observed protective effect of basil and sage supplementation on nutritional indices is consistent findings on strong antioxidant activity of phenolic compounds and essential oils from Lamiaceae herbs. The extent of this protection depends not only on the herb type and dosage, but also on the specific fatty acid composition and natural antioxidant content of each oil, which supports the variable responses observed among oil types in this study.

Total low molecular weight antioxidant activity

The assessment of antioxidant activity is another important aspect in the evaluation of oil quality. This bioactive property is related to the lipid composition and the presence of natural antioxidants in the oil1. Natural antioxidants are extracted from natural sources and their extracts, including rosemary, thyme, garlic, coffee beans, catnip, roselle seed, sage, kenaf seed, potato peel and sugar beet pulp, spices and herbs46,91. A significant body of research has been conducted on the utilization of natural antioxidants extracted from plant sources in edible oils92. One of the reasons for this is that natural antioxidants demonstrate superior antioxidant and heat stability compared with synthetic antioxidants in a range of edible oils92. Moreover, natural antioxidants offer a number of advantages over their synthetic counterparts. These include high consumer acceptability, safety, high antioxidant capacity, potential health benefits and the absence of the need for safety testing in accordance with legislative requirements93.

The total low molecular weight antioxidant activity (TAA) was examined in control oils cold-pressed from the seeds and berries of eight plant species, as well as macerates prepared with the addition of herbs (sage or basil leaves in doses of 50–100 g), after 10 months of storage (Fig. 3a). The results were compared with pre-storage TAA values previously published by Laskoś et al.21 (Fig. 3b). The addition of herbs showed a beneficial impact on the TAA of oils stored for 10 months (Fig. 3a). The positive effect of herbal additives on the TAA content in cold-pressed oils, initially reported by Laskoś et al21., persisted throughout the storage period. This was evidenced by significantly higher TAA in all macerates compared with their corresponding control oils (Fig. 3a). This effect was particularly pronounced in sage macerates of seed oils from BC, EP, H, W, R, and SB-R. A similar effect was observed in BC and S seed oil macerates with 100 g of basil (Fig. 3a). The observed enhancement of TAA can be attributed to the bioactive compounds present in the added herbs. In basil, the main compounds responsible for antioxidant activity are phenolic acids, such as rosmarinic acid, and terpenoids including eugenol and linalool75. According to the same study, sage contains several major antioxidant compounds, including rosmarinic acid, carnosic acid, carnosol, and rosmanol. Notably, the lipid-soluble compounds carnosic acid and carnosol – characteristic of Lamiaceae family plants such as rosemary and sage – are well known for their strong antioxidant properties. Carnosic acid shows high reactivity toward reactive oxygen species (ROS), undergoing oxidation to form various metabolites, including carnosol. In this way, carnosic acid acts as a ROS scavenger, neutralizing toxic ROS through its own oxidation. Carnosol, on the other hand, primarily exerts its antioxidant effect by inhibiting lipid peroxidation73. In contrast to carnosic acid, the direct radical-quenching capacity of carnosol is relatively weak. Instead, carnosol is thought to interact directly with lipid radicals, thereby interrupting and blocking the lipid peroxidation chain reaction73.

Fig. 3
figure 3

(a) The total low molecular weight antioxidant activity (TAA) of cold-pressed oils and macerates after 10 months of storage. Analysed oils: black cumin (BC) seed oil, safflower (S) seed oil, borage (B) seed oil, evening primrose (EP) seed oil, hazelnut (H) oil, walnut (W) oil, sea buckthorn-rapeseed (SB-R) oil and rapeseed (R) oil. Control oil—cold-pressed oil without any additives. Macerate – oil obtained from the maceration of 50–100 g of leaves from either sage (Salvia officinalis L.) or basil (Ocimum basilicum L.). Different letters indicate significant differences according to Duncan’s test (p ≤ 0.05), analysed separately for each species. (b) Relative TAA after storage, expressed as percentages. Values represent TAA in control oils and macerates after 10 months of storage compared with their initial TAA values reported in Laskoś et al. (2021). Initial TAA values (before storage) were set to 100%, indicated by the horizontal black line.

Full size image

The BC macerates showed a more pronounced decline in TAA following storage, contrasting with the observed pattern in other oils (Fig. 3b). However, the reduction was less pronounced when comparing the control BC oil with the 100 g herb dose macerates than with the 50 g dose (23–29% and 35–37% reductions, respectively) (Fig. 3b). Among the BC oil macerates, the smallest TAA decrease following storage was observed with the 100 g basil dose (Fig. 3b). After storage, the macerates of the remaining oils showed either a smaller reduction in TAA or only slight differences in TAA reduction compared with their respective control oils (Fig. 3b). The EP, W, and SB-R oil macerates demonstrated a clear trend of smaller reductions in TAA in macerates with the 100 g herb dose. However, in the SB-R macerates the dose effect was less pronounced than in EP or W oils (Fig. 3b). The EP, W, and SB-R oil macerates with 100 g doses of sage and basil showed TAA reductions of 45–56%, 40–47%, and 44–46%, respectively (Fig. 3b). Higher reductions were observed with the 50 g dose: 64–68%, 53–63%, and 51%, respectively (Fig. 3b). Additionally, in the EP and W oil macerates showed greater reductions with basil additives than with sage additives, particularly with 100 g doses (Fig. 3b). The S seed oil macerates exhibited a smaller TAA reduction after storage, ranging from 52% to 58%, compared with a 70% reduction in the control oil (Fig. 3b). However, the addition of 50 g of basil showed no differences compared with the control oil (Fig. 3b). The B seed oil macerates demonstrated a less pronounced TAA reduction after storage compared with the control oil, ranging from 45% to 49%, regardless of herb species or dose (Fig. 3b). For the H and R oil macerates, the storage-induced TAA reduction was significantly lower than in the control oil only with the sage dose, while in the basil dose TAA showed similar reduction levels in the control oil (Fig. 3b). Following storage, the addition of 100 g of sage resulted in a 40% TAA reduction in the H oil macerates, while a significantly greater reduction (50%) was observed with the 50 g dose (Fig. 3b). In contrast, the R oil macerates with sage showed a comparable TAA reduction of 47–49%, regardless of the dose (Fig. 3b).

Storage conditions significantly influence the level of oxidative changes and antioxidant activity35, as demonstrated by the findings of our current study (Fig. 3). After a 10-month storage period, we observed a decrease in TAA values across all oils. The addition of herbs to the oils had a significant positive effect on the TAA during storage, resulting in less reduction in TAA compared with control oils, with the exception of BC oil macerates. As shown in Fig. 3b, the TAA values of S, B, EP, H, W, SB-R, and R control oils decreased substantially, ranging from 54% to 78% compared with their corresponding pre-storage values. The control BC oil, however, showed a relatively modest reduction of 18%. In the macerates, the post-storage TAA reduction was significantly lower than in the control oils, ranging from 40% to 69% for S, B, EP, H, W, SB-R, and R oils, and from 23% to 37% for BC oil (Fig. 3b). The least reduction in TAA after storage was observed in sage macerates (100 g) from EP, H, W, SB-R and R oils. (Fig. 3b). These findings align with existing literature21,43 demonstrating that oil chemical composition significantly impacts its antioxidant capacity. These findings also support the theory that the highly lipophilic antioxidant constituents of sage leaves, such as carnosic acid and carnosol73, especially at higher leaf doses, confer sustained high antioxidant activity during prolonged storage of oils. Although this study primarily focused on fatty acid composition and antioxidant activity, a more complete assessment of oil quality would benefit from including additional parameters such as peroxide value, acid value, total phenolic content, and tocopherol levels. Future research should also explore the optimisation of maceration conditions—such as herb concentration, duration, and temperature—and investigate the impact of long-term storage on oil quality. The application of advanced analytical methods, including HPLC, GC-MS, and lipidomics, could provide a more detailed understanding of the release and stability of bioactive compounds, as well as the oxidative degradation pathways occurring during storage. This would enhance insight into the antioxidant mechanisms and functional potential of herb-macerated oils.

Conclusions

To meet increasing consumer nutritional expectations, chemical analyses were conducted on eight cold-pressed base oils derived from oilseeds with favourable fatty acid compositions: black cumin (Nigella sativa L.), borage (Borago officinalis L.), evening primrose (Oenothera biennis L.), safflower (Carthamus tinctorius L.), walnut (Juglans regia L.), common hazel (Corylus avellana L.), oilseed rape (Brassica napus L.), and sea buckthorn (Hippophae rhamnoides L.). Additionally, macerates were prepared by the addition of basil or sage herbs to these oils. All samples were analysed both before and after 10 months of storage. Each vegetable oil maintained a distinct fatty acid profile based on its plant origin, with varying effects on specific fatty acids or groups. The analysis revealed a high proportion of unsaturated fatty acids in all oils. Linoleic acid, which is beneficial for human nutrition, was the predominant fatty acid in BC, S, B, EP and W oils. In contrast, monounsaturated oleic acid dominated in SB-R, R, and H oils. The probable incorporation of basil and sage antioxidants, including carnosic acid, carnosol and rosmarinic acid, into cold-pressed oils could explain moderate alternation to the fatty acid profile in macerates. These compounds may help maintain favourable fatty acid proportions during slow screw pressing. The cold-pressing method of affects the oxidative stability of the final product, thus cold-pressed oils contain more pro-oxidant components than refined oils. Also, the fatty acid profile alternations could be determined by the oil matrix and its initial antioxidant capacity. However, these changes were relatively minor compared with the base oil composition. Basil and sage supplementation contributed to maintaining more favourable fatty acid proportions in case of black cumin seed oil and sea-buckthorn-rapeseed oil, particularly the proportion of linoleic acid, suggesting selective stabilization of oxidation-prone PUFAs. Also, in black cumin seed oil, herb incorporation increased the proportion of PUFAs and enhanced nutritional indices. This oil showed the most significant effects of herbal additives. This protective effect od herbal additives is likely mediated by exogenous herb-derived phenolics and terpenoids.

Long-term refrigerated storage led to variable reductions in total low molecular weight antioxidant activity across the oils. Herb maceration consistently mitigated the loss of antioxidant activity during storage, with sage generally providing stronger preservation than basil, particularly at higher doses (100 g). This phenomenon could be attributed to bioactive compounds in basil, including phenolic acids (e.g., rosmarinic acid) and terpenoids (e.g., eugenol, linalool), as well as lipophilic antioxidants in sage, such as carnosic acid and carnosol, which act via reactive oxygen species scavenging and inhibition of lipid peroxidation. These findings demonstrate that herbal maceration significantly preserves the antioxidant capacity of cold-pressed oils during long-term storage, highlighting the role of exogenous bioactive compounds in stabilizing lipids.

All tested oils and macerates showed potential benefits for human health. This study provides valuable insights into maceration and storage effects on fatty acid profiles of unconventional cold-pressed oils. Additionally, the cold-pressed oils and their macerates from unconventional oils may prove valuable in the development of functional foods due to their favourable fatty acid profiles and nutritional indices. These findings indicate that strategic incorporation of antioxidant-rich herbs can improve both the nutritional quality and shelf-life stability of cold-pressed oils, offering a practical approach to producing functionally enriched edible oils. Further research is required to optimise herb maceration length for enhanced nutritional value and to evaluate oxidative stability, for example by measuring the formation of hydroperoxides during storage. Future studies should also incorporate additional oil quality markers and advanced analytical techniques to better understand oxidative stability, bioactive compound dynamics, and the functional mechanisms of herb-macerated oils during long-term storage. The variations in fatty acid composition and nutritional indices suggest commercial potential for these oils in cosmetics, nutraceuticals and health-focused food formulations.

Materials and methods

Experimental design and sampling

In the first step of the experiment, crude (base) oils (control) were obtained by pressing raw materials on a slow-speed screw press (Farmet, UNO 1,1 kW, Czech Republic) at a temperature ranging from 33 to 35 °C. The raw materials included eight species: black cumin (BC; Nigella sativa L.), borage (B; Borago officinalis L.), common evening primrose (EP; Oenothera biennis L.), safflower (S; Carthamus tinctorius L.), walnut (W; Juglans regia L.), common hazel (H; Corylus avellana L.), oilseed rape (R; Brassica napus L.), and sea buckthorn (SB; Hippophae rhamnoides L.). Seeds were the raw material in the case of the first seven species. For sea buckthorn, berries served as the raw material and were initially macerated in rapeseed oil (proportion 1:1), resulting in SB-R oil. Raw materials of all species were purchased from a certified source, providing evidence of origin and variety (RAFPAK S.C., Tyczyn, Poland and Vege Market Majriusz Rajczyk, Kraków, Poland). Cold-pressing was followed by immediate gravity filtration, gradual cooling to a temperature of about 15 °C, and finally bottling.

The next step of the experiment was the production of macerates. To all crude cold-pressed base oils, herbal material – sage (Salvia officinalis L.) and basil (Ocimum basilicum L.) – was added in the form of whole leaves in doses of 50–100 g. An individual macerate was a combination of base oil type, the species of the added herb, and its dose. To eliminate any pathogen damage or morphological alterations, quality control of the raw material was performed. Afterwards, whole leaves of herbs in appropriate doses (50–100 g) were placed in glass vessels, to which 250 mL of base oil was then added. The maceration lasted for 10 days and took place in darkness at a constant temperature of 15 °C. The maceration process was completed by separating the macerates from the plant tissue through decantation into dark glass bottles. Samples of both base oils (controls) and all obtained macerates were taken to analyse fatty acid composition.

In the last step of the experiment base oils and macerates were stored for 10 months in a refrigerator at 8–10 °C. To check the stability of the fatty acid composition of the base oils and macerates, samples were taken for the second time after 10-month storage under refrigeration. In total, 10 treatments of 8 species were analysed – 64 individual macerates and 16 control oils. Each treatment was analysed in three replications. The scheme of the experimental design is provided in Fig. 4.

Fig. 4
figure 4

A schematic of the experimental design, including the preparation of macerates, the raw and herbal materials, and the sampling for analyses.

Full size image

Qualitative and quantitative determination of fatty acids composition

Fatty acid profiles were determined by gas chromatography following derivatization to fatty acid methyl esters (FAMEs) (Fig. S3). All chemicals were purchased from Merck (Darmstadt, Germany). Briefly, 5 µl oil sample was dissolved in 2 ml chloroform and 2 ml 20% sulphuric acid (VI) solution in methanol was added. The mixture was heated at 100 °C for 1 h and then cooled down. Afterwards, 1 ml water was added and vigorously mixed. When the two layers separated, the lower, chloroformic one was collected and filtered through cotton. Samples prepared in this manner were analysed with an Agilent Technologies 7820 A gas chromatograph with flame ionization detector (FID); the column used was HP-88 60 m x 0,25 mm x 0,20 μm, the gas used was hydrogen, temperature gradient 120–250 °C in 15 min. A FAMEs standard (SUPELCO 37 COMPONENT FAME MIX) was used to identify and quantify FAMEs. The results were calculated as % content of each fatty acid in the sample mass.

Nutritional indices

The following nutritional indices were analysed in the cold-pressed oils: ω−6/ω−3 – the ratio of ω-6 (C18:3n-6, C18:2n-6,9, C18:2n-6) and ω-3 (C18:3n-3) fatty acids; AI – atherogenicity index; TI – thrombogenicity index; HH – hypocholesterolemic/hypercholesterolemic ratio; PUFAs/SFAs – the ratio of the total percentage content of polyunsaturated and saturated fatty acids.The AI and TI indices, and the HH ratio were calculated according to the following equations:89,94

$$AI = [C12:0 + (4 \times C14:0) + C16:0] / \Sigma UFA]$$
(1)
$$\begin{aligned}TI &= \left( {C14:0 + C16:0 + C18:0} \right)/[(0.{\text{5 }} \times \Sigma MUFA) + (0.{\text{5 }} \times \Sigma n - 6~PUFA) \\&\quad+ ({\text{3 }} \times \Sigma n - 3~PUFA) + \left( {n - 3/~n - 6} \right)]\end{aligned}$$
(2)
$$HH = (cis - C18:1 + \Sigma PUFA)/\left( {C12:0 + C14:0 + C16:0} \right)$$
(3)

Total low molecular weight antioxidant activity

The total low molecular weight antioxidant activity (TAA) of cold-pressed oils and macerates stored for 10 months was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, as described by Laskoś et al.21. The basic DPPH protocol95 was adapted to 96-well microtitre plates and to the measurement of absorbance by ELISA reader. A solution of 0.5 mM of stable free radical DPPH (SIGMA) in methanol was used. Absorbance was determined after 30 min of the reaction at 37 °C at 515 nm using reader Model 680 (Bio-Rad Laboratories, Hercules, CA, USA). Trolox was used as the standard in the following eight concentrations: 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, and 3 nM. Each concentration was pipetted (17.5 µL) into three wells and then 250 µL of 0.5 mM DPPH solution was added on each microtiter plate. Trolox equivalents for each measurement were calculated using the equation of linear regression from the calibration curve presenting linear relationships between absorbance and different Trolox concentrations. The results were expressed as µmoles of Trolox equivalents per 1 g of oil or macerate. For each oil and macerate, two 200 µL samples were pipetted into 1.5 mL vials and their weight was determined. Next, after adding 500 µL absolute ethanol (EMPLURA Ethanol absolute, Merck), the samples were shaken for 2 h at room temperature and in the dark on a Yellow Line OS 5 Basic shaker (560 cycles/min). Next, after phase separation, each sample was measured two times on separate plates by pipetting 50 µL from the upper alcoholic phase and then 250 µL of 0.5 mM DPPH solution into 3 wells of the microtiter plate.

Statistical analysis

The data were analysed using three software packages: Statistica 13 (version 14.1.0.4, StatSoft, Inc., Tulsa, OK, USA), Past software 4.06b96 and RStudio (version 2024.04.2 + 764, RStudio Team, PBC, Boston, MA, United States). The means (n = 3) obtained for each treatment were subjected to a two-way ANOVA and post-hoc Duncan test (p ≤ 0.05) in Statistica 13. Heatmap and cluster analysis were conducted using the pheatmap function in RStudio. Principal component analysis was performed as a function included in Past 4.06b.