2.2.1 Moisture and protein content

While the percentage of moisture was calculated using (AOAC, 1990) analytical methods, that of protein was computed from the nitrogen content using the Kjeldahl conversion factor of N (%) × 5.3.

2.2.2 Ash, fat, fibre, and total carbohydrates contents

The total ash content was determined by the process of mineralising the sample seeds at 600 °C for 8 h. Soxhlet extraction with hexane as a solvent was used to calculate fat content with an extraction time of 8 h. Acid detergent fibre (ADL) were determined according to the methods of Van Soest et al. (1991). The total carbohydrates (TC) were measured as follows: TC = 100 minus moisture, protein, ash, fibre and fat percentages altogether.

2.6.1 Tocopherol composition

The NF EN ISO 9936:2006 method was used to determine tocopherol composition. An amount of 0.5 g of the seed oil was diluted with 5 mL of hexane, and a 5 μL sample was injected. The sample was analysed using Agilent 1100 HPLC (CA, USA) with a G 1354 quaternary pump, a G 1313 A standard autosampler, a G 13211 fluorescence detector set to λ excitation = 295 nm, and λ emission = 330 nm and a Chemstation software. A normal-phase column (hypersil silica, Hewlett Packard), with dimensions 250 mm × 4.6 mm × 5 μm, was used with hexane / isopropanol (99.5: 0.5, v/v) as a mobile phase. The system was isocratically operated at a flow of 0.5 mL /min. Separations were done at 30 °C and the quantification was based on an external standard method. Tocopherol standards mixed in hexane solution (2 mg/mL) were prepared based on α-, β-, γ-, and the δ- tocopherols.

2.6.2 Sterol analysis (ST)

NF EN ISO 12228 method (1999) was used for sterol separation. Lepidium sativum seed oil (250 mg) was refluxed during 15 min with a 5 mL ethanolic KOH solution (3%, w/v). A quantity of 1 mg of FLUKA cholesterol was added as an internal standard with a few anti-bumping granules. The compound was diluted at once using ethanol (5 mL). The unsaponifiable part was eluted through a glass column filled with an aluminium oxide slurry in ethanol (1:2, w/v) with 5 mL of ethanol and 30 mL of diethyl ether at a flow of 2 mL/min. A rotary evaporator was used to evaporate the solvent at 40 °C under reduced pressure. Ether was then completely evaporated under a nitrogen stream. Sterols were determined through a FLUKA silica gel F 254 plate which was developed in the solvent system n-hexane/diethyl ether (1:1, v/v). Similarly, for the detection of sterols, methanol was sprinkled over the thin-layer plate. Sterol bands were scratched from the plate and extracted with diethyl ether. Trimethylsilyl ether sterol derivatives (TMS) were prepared in a glass vial screw cap by adding up 100 µL of a silylating reagent, namely N-methyl-N-(trimethylsilyl) trifluoroacetamide/pyridine (1:10, v/v). The TMS were subsequently heated to 105 °C during 15 min.

Mixtures of sterol standard solutions were derivatized. The eluted compounds were cholesterol, sitosterol, stigmasterol, ergosterol and campesterol. The TMS sterol derivatives analysis was carried out using an Agilent 6890 N GC (CA, USA) equipped with an FID and the GC Chemstation software. An HP-5 ms GC column was used (0.32 mm i.d. × 30 m in length and 0.25 µm in film thickness, CA, USA). Helium (He) was used as a carrier gas with a flow of 1.99 mL/min in the split/splitless injector with a split ratio of 1:200. With respect to the detection and injection temperatures, they were set to 320 °C with an injection volume of 1 µL. Aiming to guarantee a maximal elution of all STs, all analyses were set to 71 min. It is trusty to note that the used parameters are as follows: Injection temperature was at 320 °C; Column temperature: a gradient of 4 °C/min from 240 °C to 255 °C; Sterol peaks were determined following the NF EN ISO 12228 (1999) method.

2.6.3 Total polyphenol content determination (TPC)

The identification of phenolic compounds was conducted through a 2-time extraction of an oil solution in hexane, whereby 5 g of oil was added to 10 mL of hexane along with a 10 mL methanol/water mixture (80:20, v/v). A rotary evaporator was used to remove the methanolic content at 45 °C. The obtained extract was then reduced to a concentration of 20 mg/mL in methanol. An amount of 100 µL of the 2 N Folin-Ciocalteu phenol reagent (Merck Schuchardt OHG, Hohenbrunn, Germany) was mixed with a 20 µL of the resultant extract and 80 µL of a saturated sodium carbonate solution (16%, w/v) was further added to the reaction compound in 5 min, precisely. Wavelength was set to 760 nm and assessed against a blank solution with a Multiskan GO microplate reader for 2 h. Each value was determined as mg of gallic acid equivalents (GAE) per g oil (Vazquez-Roncero et al., 1973; Gutfinger, 1981).

2.6.4 Total flavonoid content determination (TFC)

The whole flavonoid content was calculated in conformity with the Dewanto et al. method (2002). Twenty microliters of the methanolic extract (obtained as previously described in 2.6.3) was added to 16% of NaNO₂ in 20 µL. Six minutes later, 40 µL of 10% AlCl₃ and 100 µL of NaOH (5 M) were added. The resultant volume was set to 200 µL and mixed with distilled water. A wavelength of 510 nm was used for the absorbance reading against a blank reading with a Multiskan GO microplate reader. The total flavonoid content of the Lepidium sativum seed oil was measured as mg catechin equivalents per gram of oil (mg CE/g of oil).

2.7.1 Antimicrobial activity

The antibacterial activity of Lepidium sativum seed oil at 200 mg/mL, 500 mg/mL, and 1000 mg/mL in pure Dimethylsulfoxide (DMSO) was assessed towards four Gram-positive strains: Staphylococcus aureus ATCC 25923, Bacillus Cereus ATCC 11778, Staphylococcus aureus ATCC 6538 and Enterococcus feacium 19434 and four Gram-negative strains: Listeria monocytogenes ATCC 15313, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumonia ATCC 35657. The antifungal activity was determined against one fungal species: Candida albicans ATCC 10231.

The agar diffusion disc technique was used according to Rίos and Recio (2005). A suspension of the tested microorganisms was spread on the relevant solid media plates (Luria-Bertani) and incubated at 37 °C overnight. A day later, 4–5 loops of pure colonies were transferred to sterile saline solutions in a test tube for each bacterial strain and adjusted according to the 0.5 McFarland turbidity standard (∼10⁸ cells/mL). Sterile cotton was dipped into the bacterial suspension and the agar plates were streaked three times, each time turning the plate at a 60° angle, and finally rubbing the swab through the edge of the plate. Sterile paper discs (Glass Microfibre filters, Whatman; 6 mm in diameter) were placed onto inoculated plates and impregnated with the diluted solutions (10 µL). While Ampicillin (10 μg/disc) was used as positive control for all strains except Candida albicans for which Nystatin (100 μg/disc) was employed, the same solvent (pure DMSO) applied for the dilution of the Lepidium sativum seed oil was used as a negative control in similar conditions. Inoculated plates with discs were placed in a 37 °C incubator. Within 24 h of incubation, the results were assessed by measuring the growth inhibition zones surrounding the discs. The antimicrobial activity was then detected by the occurrence of clear inhibition zones around the discs. It should be noted that the test was conducted in triplicate.

2.7.2 Anti-inflammatory activity measurement

The anti-inflammatory effect of Lepidium sativum seed oil was assessed in-vitro through the RAW 264.7 murine macrophage cell line (ATCC) by nitrite accumulation. Cell growth was performed through 24-well plates at a density of 2 10⁵ cells/mL for 24 h at 37 °C before being treated with various concentrations of Lepidium sativum seed oil dissolved in the DMSO or the positive control (L-NAME) for 1 h. The final concentration of solvent in the culture medium was maintained at 0.1% (v/v) to avoid solvent toxicity. Lipopolysaccharide (LPS) (100 µg/mL) was added to the treatment group of plates, while medium or LPS alone was added to the control group. Following a 24 h LPS stimulation at 37 °C under 5% CO₂, the cell free supernatants were collected and Griess's reagent (1% sulfanilamide, 5% phosphoric acid and 0.1 % N-(1-naphthyl)-ethylene diamine dihydrochloride) was used to assess nitric oxide (NO) levels (Green et al., 1990). The resazurin assay protocol was used for checking the cytotoxicity of samples on cells. For the assessed concentrations (0–300 µg/mL) of Lepidium sativum sample, we found no significant cytotoxic effect. It is to worthy to note that the nitrite concentration in the samples was calculated using a sodium nitrite standard curve, and absorbance was measured at 540 nm.

2.7.3 Antioxidant activity

The antioxidant activity of Lepidium sativum seed oil was evaluated through DPPH and ABTS assays. For the DPPH assay, a Lepidium sativum seed oil sample was tested for its scavenging ability against the stable 1,1- diphenyl-2-picrylhydrazyl (DPPH) radicals (Kalantzakis et al., 2006). One millilitre of a compound of 0.5 g oil in a 5 mL of ethyl acetate was mixed with 4 mL of a 10⁻⁴ M ethyl acetate DPPH solution within a 10 mL screw cap test tube. The reaction mixture was then vortexed intensely for about 10 s. Steady-state stability was reached by keeping the tube in total darkness for exactly 30 min. Absorbance was subsequently measured at 515 nm against a blank solution. In the meantime, 1 mL of ethyl acetate was mixed with 4 mL DPPH solution for the sake of preparing and evenly measuring a control sample solution.

DPPH Radical Scavenging Activity (RSA) was determined as the DPPH concentration reduction% of the Lepidium sativum components and was calculated according to the following equation:

% [DPPH]_red = 100 × (1 − [DPPH]₃₀ /[DPPH]₀), where [DPPH]₀ and [DPPH]₃₀, using DPPH concentrations in the control sample (t = 0) and in the test mixture after the 30 min reaction, respectively.

The ABTS decolorization assay was applied to measure the free-radical scavenging capacity (Re et al., 1999) with some minor adjustments. ABTS was dissolved in water to obtain a concentration of 7 mM. The ABTS^•+ radical was generated by reacting a 140 mM K₂S₂O₈ solution with this stock solution, leaving the mixture in the dark at room temperature for 12–16 h before use. ABTS^•+ solution was diluted with methanol to reach an absorbance of 0.7 ± 0.02 at 730 nm. After a 2-min incubation in a glass cuvette at 30 °C, absorbance readings were taken after adding an adequate volume of the Lepidium sativum seed oil sample, with methanol as a blank, or Trolox standard, to 1 mL of diluted ABTS^•+ solution. A decrease in absorbance was obtained at 734 nm. All the measurements were carried out in triplicate. The free radical scavenging capacity was calculated as ABTS^•+ inhibition percentage of the Lepidium sativum seed oil sample against a Trolox standard curve (prepared at different concentrations in the range of 40–500 mM of Trolox). The results were described as mM of Trolox equivalent antioxidant capacity (TEAC, mM).

2.9.1 Fourier transform infrared spectroscopy (FTIR) analysis

FTIR analytical technique was used to identify the functional groups found in the Lepidium sativum seed oil sample using a Thermo Scientific Nicolet 380 FTIR spectrophotometer. The FTIR spectra recorded in the absorption band mode ranged from 500 to 4000 cm⁻¹ with a spectral resolution of 4 cm⁻¹ at 32 scans. It should be noted that Lepidium sativum seed oil was analysed without any prior treatment. To test repeatability, analyses were conducted in triplicate and average spectra were used.

2.9.2 Thermal analysis

Thermogravimetric (TG) curves were acquired with a TGA-50 Shimadzu analyzer. Thermogravimetric analyses (TGA) were carried out in the temperature range from 25 to 600 °C using a high-purity synthetic air (0 gas) atmosphere (100 mL/min). Alumina crucibles were used with 10 °C/min as a heating rate, and 5 mg as sample mass. For data analysis, the Shimadzu TA-60WS (2.20) software was applied to simultaneously measure the thermogravimetric (TG) and first derivative thermogravimetric (DTG) curves.

2.9.3 Rheological measurement

AR 2000ex rheometer (TA Instruments, Ltd., Crawley, UK) was used to determine oil viscosity. According to the cone-plate geometry, shear rate ranged from 0.01 to 500 s⁻¹ with a temperature range of 20 up to 120 °C. What is trustworthy to mention is that temperature stability was attained using a Peltier-type connected water bath (±0.1 °C). The diameter, cone angle and the gap between the cone and plate were 40 mm, 1° and 200 µm, respectively (Chouaibi et al., 2012). Shear stress was calculated as a shear rate function and data were provided according to the Newtonian law model:

Furthermore, data related to viscosity dependence toward temperature were fitted to the following exponential models:

3.4.1 Tocopherols

Seed oils are a potential source of tocopherols that are known to be highly effective in preventing oxidative deterioration of polyunsaturated fatty acids in most plants thanks to their natural lipophilic and antioxidant properties. The tocopherol composition of Lepidium sativum seed oil obtained by HPLC is shown in Table 4. The major components were γ-tocopherol accounting for about 86.23% of the total tocopherols, followed by δ-tocopherol, β-tocopherol, and α-tocopherol. These results agree well with those found by Diwakar et al. (2010) in Lepidium sativum seed oils extracted with a hydraulic press, by Soxhlet using petroleum ether as solvent and by supercritical carbon dioxide in which γ-tocopherol represented 80.4%, 80.9%, and 80.3% of total tocopherols, respectively. In our study, γ-tocopherol content was lower than those found by Moser et al. (2009) (79.04%) in Lepidium sativum seed oil extracted by Soxhlet with hexane. With respect to other Mediterranean vegetable oils, the Lepidium sativum seed oil exhibited a higher content of γ-tocopherol. In fact, according to Zarrouk et al. (2009), the γ-tocopherol content ranges from 5 ± 1 mg/kg to 22 ± 1 mg/kg for olive oil, from 84 ± 4 mg/kg to 92 ± 7 mg/kg for Milk thistle seed oil, and from 12 ± 1 to 434 ± 7 mg/kg for argan oil. Given its ability to cross the blood brain barrier, γ-tocopherol, which is found at elevated levels in cold pressed Lepidium sativum seed oil, could be useful to attenuate the toxic effects of 7KC. Such hypothesis is confirmed by Debbabi et al. (2016) who have proven that γ-tocopherol is able to impair 7KC-induced loss mitochondrial transmembrane potential associated with increased permeability to propidium iodide. The latter is an indicator of plasma membrane damages and murine microglial BV-2 cell death. This isomer is also able to prevent the decrease in ABCD3 protein levels, which allows the evaluation of peroxisomal mass, and in mRNA levels of ABCD1 and ABCD2, encoding two peroxisomal transporters involved in peroxisomal β-oxidation.

The low content of α-tocopherol (0.31%) is in compliance with that obtained in a previous study conducted by Nehdi et al. (2019) on Lepidium sativum seed oil, in which this isomer represented 0.45% of total tocopherols. Furthermore, α-tocopherol was found in higher amounts in Lepidium sativum seed oil extracted by Soxhlet using a mixture of n-hexane/2-propanol (3:1, v/v) (12.3%) (Zia-Ul-Haq et al., 2012). It is worth mentioning that β-tocopherol was not detected in Lepidium sativum seed oil extracted by a hydraulic press, by Soxhlet and by supercritical carbon dioxide (Moser et al., 2009; Diwakar et al., 2010). According to Fatnassi et al. (2009), when compared to other tocopherols, the α-tocopherol is highly approved for both humans and animals thanks to its higher biological activity, yet γ-tocopherol shows a higher antioxidant capacity than α-tocopherol. Total tocopherol content in Lepidium sativum seed oil (136.83 ± 7.6 mg/100 g) is higher than those reported by Diwakar et al. (2010) but lower than that found by Moser et al. (2009) (179.9 ± 0.9 mg/100 g).

3.4.2 Sterols

Table 4 illustrates sterol composition of Lepidium sativum seed oil obtained by GC-FID. Thirteen phytosterols were identified: cholesterol, 24-methylenecholesterol, stigmasterol, sitosterol, sitostanol, campesterol, campestanol, clerosterol, Δ7-campesterol, Δ 5,24-stigmastadienol, Δ5-avenasterol, Δ 7-avenasterol and Δ7-stigmastenol. The β-sitosterol accounted for 42.57 mg/100 g and had the highest sterol content, followed by the campesterol (20.04 mg/100 g), and the Δ 5,24- stigmastadienol (11.82 mg/100 g). Thus, β-sitosterol, campesterol, and Δ 5,24- stigmastadienol were the major sterols, and together they made up 90.21% of Lepidium sativum seed oil. It is noteworthy to say that β-sitosterol was also the sterol marker in Lepidium sativum seed oil extracted by Soxhlet with hexane and represented only 40.38% of total sterols. This phytosterol was followed in descending order by campesterol, avenasterol, cholesterol, stigmasterol, dihydrolanosterol, and β-amyrin. Cholesterol, sitosterol, and stigmasterol contents were higher than those found in the present study (Moser et al., 2009).

The Lepidium sativum seed oil overall phytosterol content (0.825 mg/g) was lower than those cited by Zarrouk et al. (2019) in olive oil (1726 ± 45 mg/kg to 2936 ± 59 mg/kg), Milk thistle seed oil (5088 ± 71 mg/kg to 5891 ± 118 mg/kg), and argan oil (1881 ± 25 mg/kg to 2330 ± 79 mg/kg). Along with the effect of the intake of phytosterols on the development of neurodegenerative diseases, these bioactive nutrients have been recently reported to possess anti-neurodegenerative effects (Cheng et al., 2014; Dierckx et al., 2019). This has been confirmed by treating neuroinflammation which is a common pathological feature of many neurodegenerative diseases, namely Alzheimer and Parkinson (Ransohoff, 2016; Russo and McGavern, 2016; Subhramanyam et al., 2019).

It is interesting to note that brassicasterol is absent in Lepidium sativum seed oil, which typically comprises 5–20% of phytosterols in oils from other plants in the Brassicaceae family, such as rapeseed, canola, and mustard oils (Kochhar, 1983; Phillips et al., 2002).

3.4.3 Total polyphenol and flavonoid contents

Thanks to their diverse chemical structures, phenolic phytochemicals have the potential to protect cellular components against oxidation (Bahloul et al., 2014). In the same way, polyphenols are known for not only their potential health virtues, but also their capacity to prevent several chronic diseases, such as cancer and high blood pressure (Liara et al., 2013). It has also been reported that naturally synthesized polyphenols contribute to the effective fighting of neurodegenerative disorders such as brain aging (Sarubbo et al., 2018). So far, no previous studies have investigated the total polyphenol and flavonoid contents of Lepidium sativum seed oil. The total polyphenol content (TPC) of Lepidium sativum seed oil obtained by the Folin-Ciocalteu method was of 0.23 ± 0.00 mg GAE/g. Zia-Ul-Haq et al. (2012) and Kasabe et al. (2012) have reported TPCs of 120.26 ± 1.52 mg CE/g and 21 mg GAE/g, respectively, in the methanolic extract of Lepidium sativum seeds. Ait-yahia et al. (2018) have also reported a TPC of 1.629 ± 0.13 mg GAE/g in n-butanol Lepidium sativum seed extract.

According to Ismail et al. (2010), flavonoids are the most common and most widely distributed group of plant phenolic compounds, which are usually considered as very effective antioxidants. The total flavonoid content of Lepidium sativum seed oil obtained according to Dewanto et al. method (2002) was of 0.78 ± 0.02 mg CE/g. This value was higher than that found by Różańska et al. (2018) when investigating extra virgin olive oil (26.6 ± 13.5 mg/kg).

3.5.1 Antimicrobial activity

The antimicrobial activity of the Lepidium sativum seed oil was assessed by the agar diffusion disc technique according to Rίos and Recio (2005). Our results showed that the solvent (pure DMSO) employed in the dilution of the Lepidium sativum seed oil did not have any antibacterial capacity against all tested strains. Likewise, Lepidium sativum seed oil had no inhibitory effect on all tested strains. Such a result is in agreement with that found in the research work of Getahun et al. (2020) who have substantiated that the seed oil of the same plant originating from India and extracted with petroleum ether in a Soxhlet apparatus have not any activity toward Gram – (Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 10031) and Gram + (Saphylococcus aureus ATCC 25923 and Bacillus cereus ATCC 14579) bacteria. However, our findings are not in accordance with those cited by Alqahtani et al. (2018) on Lepidium sativum seed oil extracted by Soxhlet using petroleum ether as solvent. In fact, the results related to the Lepidium sativum seed oil antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumonia and Candida albicans showed that the latter were susceptible to Lepidium sativum seed oil. For all mentioned bacteria and fungi, the minimum inhibitory concentration (MIC) was 47.5 mg/mL, except for Salmonella enterica that revealed a higher MIC of 90 mg/mL. The minimum bactericidal concentration of Lepidium sativum seed oil was found to be equivalent to 100 mg/mL for inhibiting the growth of all bacteria and fungi. Alqahtani et al. (2018) have confirmed the findings of Abo El-Maati et al. (2016) related to the antimicrobial activity of Lepidium sativum seed oil in terms of its spectrum against all tested microorganisms except for Staphylococcus aureus which was proven to be resistant to the used Lepidium sativum seed oil. When compared to other studies conducted on the determination of the MIC of Lepidium sativum seed oil toward bacterial and fungal strains, the variations in MIC values may be attributed to the differences in the adopted antimicrobial activity technique and the used oil extraction procedure (Alqahtani et al., 2018). The impact of the method used for the evaluation of the antimicrobial activity on the results was also consolidated by the findings of Boyanova et al. (2005).

Differences in the antimicrobial activity of Lepidium sativum seed oil is likely to emanate from the sensitivity of the strains depending on the inability of the antimicrobial agent present in the oil to diffuse uniformly into the agar. It is possible that Lepidium sativum seed oil's inefficiency is due to its low TPC. In fact, many studies have proven the positive correlation between the phenolic compounds content and the antimicrobial activity (Brahmi et al., 2020). According to Cowan (1999), polyphenols such as tannins and flavonoids, are powerful antimicrobial substances. The number and position of hydroxyl group in the aromatic ring of these compounds can also enhance their toxicity to the microorganisms. Furthermore, lipophilic flavonoids can damage microbial membranes by increasing lipid fluidity. It is worth noting that the antimicrobial activity is dependent not only on the presence of phenolic compounds, but also on the existence of numerous secondary metabolites.

3.5.2 Anti-inflammatory activity

The anti-inflammatory effect of Lepidium sativum seed oil was assessed in-vitro through the RAW 264.7 murine macrophage cell line (ATCC) by nitrite (NO) accumulation (Fig. 1). NO production was reported as a principal mediator playing an essential role in various physiological and pathophysiological processes. Meanwhile, the excess in NO may contribute to tissue injury in inflammatory diseases. Indeed, it can react with superoxide radicals to form the harmful peroxynitrite. Consequently, the prevention of NO generation may be an interesting strategy to treat various inflammatory disorders (Bourgou et al., 2010). The results illustrated in Fig. 1 are expressed as quantity of NO production in LPS-stimulated Raw 264.7 macrophages. Our results proved that LPS treatment triggered significant nitrite accumulation in control cells. Interestingly, NO production was significantly inhibited by Lepidium sativum seed oil fraction treatments. In fact, cold pressed Lepidium sativum seed oil was found to decrease NO release in a concentration-dependent manner. Such a result is in agreement with that cited by Alqahtani et al. (2018) on the anti-inflammatory activity of Lepidium sativum seed oil extracted by Soxhlet using petroleum ether as a solvent. These authors have demonstrated the presence of a concentration-dependent protection of the human red blood cell (HRBC) membrane by Lepidium sativum seed oil. The observed levels of oil protection were 21%, 11% and 7% for respective concentrations of 300, 200 and 100 µg/mL. When compared to a standard drug (diclofenac Na), the inhibition percentages of hemolysis were significantly higher, showing 65%, 63%, and 61% membrane stabilization at concentrations of 300, 200, and 100 mg/mL, respectively. When tested at 75 µg/mL, 150 µg/mL and 300 µg/mL, Lepidium sativum seed oil decreased NO production by macrophages by 56%, 66% and 69%, respectively. The IC₅₀ value was of 60 µg/mL, indicating an interesting anti-inflammatory activity of Lepidium sativum seed oil.

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Fig. 1

Effect of Lepidium sativum seed oil on LPS-induced NO production in RAW 264.7 macrophages. Different letters on the top of data bars indicate significant differences (p < 0.05) between mean values (±SD, n = 3).

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Furthermore, Bourgou et al. (2019) have reported strong anti-inflammatory activities for carvi oils extracted with 2-methyltetrahydrofuran (MeTHF) and supercritical-carbon dioxide (Sc-CO₂) with IC₅₀ values of 27 µg/mL and 28 µg/mL, respectively. The higher anti-inflammatory activity of oil was ascribed to the effect of some active compounds, namely phenolics and sterols. It has been documented that incubating inflammatory cells with total phenolic fraction or isolated compounds from olive oil reduced anti-inflammatory enzyme activities, resulting in a reduction in NO production (Aparicio-soto et al., 2014; Cardeno et al., 2014). Besides, sterols may have a protective effect on certain mediators associated with the production of inflammatory damage. In fact, β-sitosterol has been shown to reduce NO release in activated macrophages, which has been linked to iNOS activity impairment (Moreno, 2003; Montserrat-de la Paz et al., 2012).

3.5.3 Antioxidant activities

ABTS and DPPH assays were used to assess the antioxidant activity of Lepidium sativum seed oil. The ABTS test proved that the radical scavenging activity of Lepidium sativum seed oil was of 415.6 ± 40 TEAC. To the best of our knowledge, the ABTS assay has not been used before to determine the antioxidant activity of Lepidium sativum seed oil. For the DPPH assay, the Lepidium sativum seed oil showed a considerable IC₅₀ value of 10.03 ± 0.05 mg/mL. The Lepidium sativum seed oil antioxidant activity was higher when compared with those found in earlier literature reports (Alqahtani et al., 2018; Getahun et al., 2020) in which Lepidium sativum from Saudi Arabia and India were examined using the DPPH assay. In fact, the IC₅₀ values of the seed oils extracted using Soxhlet apparatus with petroleum ether as solvent were of 40 mg/mL and 23.5 mg/mL for Saudi Arabian and Indian plants, respectively. Getahun et al. (2020) have described differences in the antioxidant activity to variations in seed oil compositions. It is worth noting that the IC₅₀ value of the seed oil from the Tunisian plant is likewise lower than that found by Umesha and Naidu (2013) (23.5 mg/mL) in cold pressed Lepidium sativum seed oil. It is also well documented that the antioxidant activity is generally related to the amount and nature of phenolic compounds. In addition, an identical correlation is recorded between the flavonoid content and the DPPH assay (Brahmi et al., 2020). According to Górnaś et al. (2015), differences in the seed oils’ DPPH radical scavenging activity may also be attributed to total tocochromanol content.

3.7.1 FTIR analysis

The importance of IR spectroscopy in molecular structure identification is related to the rich information content and to the ability to assign certain absorption bands to functional groups. Most of the bands and shoulders of the spectrum in fats and oils may be accredited to specific functional groups (Bendini et al., 2007). Since triglycerides are the main components of fats and oils, the latter dominate the fat and oil spectra.

Fig. 2 depicts the Fourier-transform infrared (FT-IR) spectrum of cold pressed Lepidium sativum seed oil. The FTIR characteristic bands of the oil sample are detected in the 3010–2800, 1750–1000, and 1000–650 cm⁻¹ regions. Such a finding is consistent with previous studies conducted by Nehdi et al. (2012) and Mulla et al. (2018) on Lepidium sativum seed oil extracted by hexane in a Soxhlet apparatus and petroleum ether, respectively. A band is marked at 3008.66 cm⁻¹, indicating the C = C-H stretching (asymmetry) cis olefinic groups, while bands at 2921.68 and 2852.1 cm⁻¹ are assigned to asymmetric and symmetric stretching vibration of the aliphatic CH₂ groups, respectively. The absorption bands of Lepidium sativum seed oil at 1742.81 and 1462.9 cm⁻¹ are associated with the ester carbonyl stretching (C = O) of fatty acids and C-H bending (scissoring) of CH₂ and CH₃ groups, respectively. In addition, the band at around 1377 cm⁻¹ (1376.41 cm⁻¹) is attributed to C-H bending of CH₂ group, whereas those around 1162 cm⁻¹ (1159.1 cm⁻¹) and 1099 cm⁻¹ (1097.81 cm⁻¹) could represent the antisymmetric stretching variation of C-O. A minor band at 720.03 cm⁻¹ represents the C-H rocking of the CH₂ group.

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Fig. 2

FTIR spectrum of cold pressed Lepidium sativum seed oil at frequency range of 4000–500 cm⁻¹.

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3.7.2 Thermal analysis

The Thermogravimetric analysis (TGA) is a useful tool to investigate several physico-chemical properties related to the oil behavior with respect to oxidative stability. Fig. 3 shows the TG and DTG curves of cold pressed Lepidium sativum seed oil. The thermal oxidative degradation processes for the assessed oil occurs as mainly three consecutive steps of mass loss in the temperature range between 180 °C and 600 °C. The first step of degradation begins at 180 °C and ends at 410 °C. As can be seen from the DTG curve, an exothermal peak occurs at about 360 °C (352.06 °C). The second mass-loss step occurs from 410 °C to 500 °C and describes an exothermal peak at approximately 440 °C (436.34 °C). This peak is followed by a final exothermal mass-loss step occurring between 500 °C and 600 °C and presenting an exothermal peak at about 550 °C (543 °C). According to Santos et al. (2004), the first degradation event is due to polyunsaturated fatty acid decomposition and is considered as the most important step to determine the edible oil thermal stability order. In fact, during heating, triglycerides produce volatile compounds that are constantly removed by vapor during heating. The products are mainly formed by the thermal reactions of the unsaturated fatty acids. The second thermal degradation event is reported to monounsaturated fatty acids decomposition. The last degradation event is attributed to saturated fatty acids thermal decomposition. Ciprioti (2014) reported the final exothermal mass-loss step to the combustion of the residual carbonaceous material from the previously occurring steps. The same thermal behavior was observed for Lepidium sativum seed oil extracted by Soxhlet with hexane (Nehdi et al., 2019), for rambutan (Nephelium lappaceum L.) seed fat, and for commercial olive, soybean, rapeseed, corn, and rice oils (Santos et al., 2004). It is worthwhile to note that the temperature corresponding to 5% of Lepidium sativum seed oil mass loss was recorded at around 300 °C, which accords well with the findings of Nehdi et al. (2019) for Lepidium sativum seed oil extracted by the Soxhlet apparatus (299 °C). When compared to other edible oils examined under the same operating conditions, Lepidium sativum seed oil was proven to exhibit the highest thermal stability followed in descending order by olive (285 °C) and sunflower (277 °C) oils. Nehdi et al. (2019) attributed the Lepidium sativum seed oil high thermal stability to its high antioxidant content and the presence of long-chain monounsaturated and saturated fatty acids. Such a finding is consolidated by that of Santos et al. (2004) who states that these oils doped with artificial antioxidants are more stable than non-doped oils.

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Fig. 3

TG and DTG curve illustrations of Lepidium sativum seed oil.

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3.7.3 Rheological measurement

Lepidium sativum seed oil exhibited Newtonian behavior at shear rates ranging from 0.01 to 500 s⁻¹ at 20 °C (Fig. 4) and even at 40 °C, 60 °C, 80 °C, 100 °C, and 120 °C (data not shown). In fact, shear stress and shear rate were found to have a linear relationship. The Newtonian law model was used to describe the flow curves of the investigated Lepidium sativum seed oil sample. Based on the determination of R² value, the Newtonian model was noted to be suitable for the rheology description of Lepidium sativum seed oil regarding flow curves (R² > 0.9). The rheological parameters of this model at 20 °C are illustrated in Table 5. These results are in accordance with those previously obtained by Maskan (2003), Kim et al. (2010), Chouaibi et al. (2012), and Gharsallah et al. (2021). These researchers have ascertained that vegetable oils are Newtonian liquids possessing high viscosity thanks to the ability of their molecules to form long chains. Table 5 explains how Lepidium sativum seed oil viscosity value $\eta$ adjusted to 20 °C (58.42 ± 0.04 mPa.s) was lower than Moringa seed oil (97.11 ± 0.02 mPa.s) (Gharsallah et al., 2021) and those of olive (76.22 ± 0.02 mPa.s), Corn (63.27 ± 0.06 mPa.s), soybean (63.23 ± 0.06 mPa.s), groundnut (69.04 ± 0.03 mPa.s), jujube seed (80.51 ± 0.02 mPa.s), and rapeseed (71.52 ± 0.02 mPa.s) oils. Yet, the value was closer to the sunflower oil (59.17 ± 0.03 mPa.s) (Chouaibi et al., 2012). Kim et al. (2010) have stated that although oil viscosity is positively influenced by the oleic acid content, it is negatively affected by linoleic acid content.

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Fig. 4

Shear stress versus shear rate of Lepidium sativum seed oil at 20 °C.

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It is fair to say that a thorough understanding of the rheological properties of natural vegetable oils is important when it comes to lubrication. Fig. 5 depicts viscosity vs temperature data pertaining to the Lepidium sativum seed oil. The findings showed that the investigated Lepidium sativum seed oil underwent an exponential decrease in viscosity ( $\eta )$ with an increase in temperature ranging from 20 °C to 120 °C. A decrease in vegetable oil viscosity is observed when molecular distance increases and/or when intermolecular bonds decline due to a significant thermal motion of molecules (Chouaibi et al., 2012). Such phenomenon is fully consistent with previous research works conducted by Santos et al. (2005) and Kim et al. (2010). The temperature effect on Lepidium sativum seed oil viscosity was evaluated by applying the exponential and Arrhenius models. The models were found to fit apparent viscosity evolution against temperature with R² values of 0.97 and 0.98, respectively (Table 5). Calculating activation energy (Ea) value can be carried out by plotting ln $\eta$ vs 1/T in a graph (data not shown). In general, high activation energy suggests the viscosity - temperature stability of vegetable oils (Chouaibi et al., 2012). Table 5 shows the activation energy of the Lepidium sativum seed oil (27.7 ± 0.03 kJ/mol). Such a value surpasses the results found by Chouaibi et al. (2012) for corn, soybean, sunflower, and groundnut oils with respective values of 20.39 ± 0.01 kJ/mol, 20.21 ± 0.01 kJ/mol, 20.07 ± 0.03 kJ/mol, 20.78 kJ/mol. However, it remains lower than that found by Gharsallah et al. (2021) for the Moringa seed oil (30.14 kJ/mol).

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Fig. 5

Effect of temperature on the apparent viscosity of Lepidium sativum seed oil.

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