3.1. Profiles of Fatty Acids, Phytosterols, Tocopherols, and Polyphenols Cold-Extracted from Milk Thistle Seed Oils from Different Area of Tunisia
Milk thistle seed oil (MTSO) is known for its high level of secondary metabolites, such as phenolic acids, tocopherols, fatty acids, and phytosterols, which depend on many intrinsic (genetic) and extrinsic (environmental) parameters [60
]. In the present study, we focused on the fatty acids, phytosterols, polyphenols and tocopherols present in MTSO. Data obtained were compared with nigella seed oil which is mainly empirically known for its toxic activity.
Milk thistle seed oil (MTSO; from Tunisia—Bizerte, Sousse and Zaghouan) and nigella seed oil (NSO; from Tunisia) were analyzed. The fatty acid compositions of these oils are given in Table 1
. Linoleic acid (C18:2 n-6) was the most abundant polyunsaturated fatty acid (PUFA) detected in MTSO and NSO. The results show that the oils used in this experiment had high levels of linoleic acid. From a nutritional point of view, as for many other dietary oils, MTSO is a good source of essential fatty acids, especially linoleic acid. Another unsaturated fatty acid quantitatively high in MTSO was oleic acid (C18:1 n-9). Palmitic acid (C16:0) was the most abundant saturated fatty acid in MTSO and NSO, followed by stearic acid (C18:0). Very long-chain saturated fatty acids (C22:0 and C24:0) were found to have high amounts in MTSO. In addition to the fatty acid profiles, the composition of tocopherols is an important parameter to define the potential health benefits of vegetable oils. The tocopherol contents of the oils studied are given in Table 2
. The results show that α- and β-tocopherols were the only ones detected in cold-pressed MTSO from Bizerte and represented 47.65 mg/kg and 1.91 mg/kg, respectively. In the oils from Zaghouan and Sousse, the four tocopherols were detected with a predominance of α-tocopherol which represented 98.92% and 88.59%, respectively. The amounts of α-tocopherol in the MTSO of Zaghouan and Sousse were in the same range as those reported by El-Mallah et al. [60
] in MTSO extracted with chloroform-methanol (84.5%) and those reported by Fathi-Achachlouei and Azadmard-Damirchi [61
] for Iranian varieties of Barak, and for MTSO extracted by hexane/isopropanol (84.9% and 86.36%, respectively). The amount of α-tocopherol was greater than that reported by Parry et al. [16
] (78.78%) in cold-pressed MTSO, and by Hasanlou et al. [63
] in MTSO (69.02%) extracted by petroleum ether (Soxhlet apparatus). In the MTSO from Sousse, the high content of α-tocopherol (considered to be the most biologically active tocopherol) [64
] was followed by γ-tocopherol, β-tocopherol, and δ-tocopherol. The amounts of β- and γ-tocopherol were greater than those observed in the MTSO from Zaghouan. Thus, the cold pressed MTSO from Zaghouan is an ideal source of total α-tocopherol, while that of Sousse could serve as a source of γ-tocopherol. It is noteworthy that compared to olive and argan oils, the highest values of α-tocopherol were observed in MTSO [46
As phytosterols are components that are present in the unsaponifiable lipid fraction of plant cell membranes [64
] and are abundant in vegetable oils (nuts, cereals), it was important to determine the content and composition of the sterols in the MTSO studied (Table 3
). β-Sitosterol was the main sterol in all oils. Schottenol was also detected at a high level in MTSO and, in particular, in Zaghouan MTSO. Campesterol was determined in appreciable amounts, especially in the MTSO from Sousse. The presence of cholesterol is in agreement with the results reported by Fathi-Achachlouei and Azadmard-Damirchi [61
] as well as by Dabbour et al. [62
]. Cholesterol could be formed by demethylation of sitosterol. The synthesis of cholesterol in plants remains to be clarified [65
]. Cholesterol can be localized in the nucleus, chloroplasts, and microsomes in free or esterified form. Cholesterol is an important constituent of the leaf wall. It should be noted that high levels of cholesterol have been reported in palm, corn, and sunflower oils [66
]. Cholesterol has never been identified, even at very low levels, in olive and argan oils, whatever the geographic origin of the oils [46
The polyphenol compositions of the oils are given in Table 4
. Homovanillic acid, vanillin, p-coumaric acid, quercetin-3β-glucoside, quercetin, and apigenin were identified in MTSO. In nigella seed oil, 2,6-dihydroxybenzoic acid, homovanillic thymoquinone acid, vanillin and some amounts of chlorogenic acid, ferulic acid, quercetin, and apigenin were detected.
As many compounds present in MTSO (phytosterols, polyphenols, tocopherols) are able to prevent nerve cell dysfunction and can cross the blood–brain barrier [67
], these data reinforced our interest in evaluating the ability of MTSO to prevent the toxic effects of 7KC and 24S [68
]. So, the ability of MTSO to prevent the side effects induced by 7KC and 24S was compared to α-tocopherol (used as a positive cytoprotective control) and to nigella seed oil (supposed as potentially cytotoxic).
The prevalence of some diseases or syndromes increase with age, such as atherosclerosis, Alzheimer’s disease, age-related macular degeneration, cataract, and osteoporosis. All of these diseases involve oxidative stress, inflammation, and/or cell death processes [11
Oxysterols could play an important role in these different age-related pathologies, in particular, in the pathophysiology of the brain. Indeed, several oxysterols can induce several side effects contributing to neurodegeneration [11
The most widely-considered oxysterols potentially involved in the pathogenesis of the processes of neurodegenerative diseases are 24S [75
], which is of enzymatic origin, and 7KC resulting from cholesterol auto-oxidation [8
In 158N murine oligodendrocytes, 7KC and 24S trigger a complex mode of cell death defined as oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY), simultaneously involving oxidative stress, apoptosis, and autophagy [73
]. 7KC and 24S induce a decrease in cell proliferation, assessed by crystal violet; an alteration of mitochondrial activity, quantified with the MTT test; increased permeability of plasma membrane, revealed with propidium iodide; overproduction of reactive oxygen species, revealed by dihydroethidium staining; caspase-3 cleavage; and activation of LC3-I into LC3-II that are typical of oxiapoptophagy. In agreement with previous studies, this complex type of cell death induced by 7KC and 24S is attenuated by α-tocopherol [36
Natural products are considered effective sources for the discovery of powerful and novel therapeutic agents, particularly dietary phytochemicals (polyphenols, terpenes, carotenoids, and others). As some of them are known to protect against oxidative stress, they could be effective in preventing 7KC- and 24S-induced side effects and in preventing and/or treating 7KC- and 24S-associated diseases. At the moment, various phytochemicals (fatty acids, polyphenols, phytosterols, and tocopherols) are able to counter the side effects of oxysterols in different age-related pathologies [68
]. Some of these phytochemicals, either food extracts or pure compounds, are present in Mediterranean diet products, such as olive oil, fruits, and vegetables.
In this context, it was interesting (i) to establish the chemical profile of MTSO from different areas of Tunisia (Zaghouan, Bizerte and Sousse) in phytosterols, tocopherols, and polyphenols, since these compounds are potentially neuroprotective, (ii) to determine their antioxidant characteristics, and (iii) to precisely determine their cytoprotective effects on nerve cells.
Currently, oxidative stress and mitochondrial dysfunction are considered key events in several degenerative diseases [77
]. Interestingly, the chemical profile of MTSO reveals the presence of numerous compounds that can prevent oxidative stress and mitochondrial dysfunction. Thus, MTSO contains high levels of antioxidant molecules (tocopherols, polyphenols) capable of reducing the overproduction of reactive oxygen species (ROS: ROO•, RO•) [78
], which can induce lipid peroxidation leading to protein carbonylation and several types of cellular dysfunction that may contribute to neurodegeneration [79
]. The presence of these compounds is consistent with the antioxidant properties of MTSO determined with the KRL, DPPH, and FRAP tests. In addition, MTSO is also rich in oleic acid (C18:1 n-9). Although (C18:1 n-9) is not an antioxidant [47
], it has been shown in 7KC-treated murine microglia BV-2 cells that this fatty acid is able to attenuate the overproduction of ROS [43
]. In addition, oleic acid as well as α- and γ-tocopherol have been shown to prevent mitochondrial, lysosomal, and peroxisomal dysfunction in different types of nerve cells [33
]. As these compounds, which have been shown to inhibit 7KC- and 24S-induced neurotoxicity are present at low levels in MTSO (the final concentrations of oleic acid, and α- and γ-tocopherol were more than one hundred-fold lower than those in the culture medium when these compounds were used alone), it is possible that some of them may act in synergy to exert cytoprotective effects and/or that several other compounds of MTSO (polyphenolic compounds, sylimarine, etc.), could be involved in the beneficial effects. We must also consider that the different compounds present in MTSO could activate and suppress several signaling pathways contributing to cytoprotection.
It should be noted that when 7KC was used at 25 and 50 μM, two concentrations capable of inducing oxidative stress and favoring oxiapoptophagy [38
], MTSO was able to attenuate the toxicity induced by 7KC (25–50 μM). As observed with α- and γ-tocopherol [47
], docosahexahenoic acid [38
], acid oleic [46
] and dimethylfumarate [72
], and argan oil [47
], MTSO (0.1% v
) was able to counteract the inhibition of cell growth associated with 7KC-induced loss of cell adhesion.
These results were highlighted by the crystal violet and the MTT assays. As observed with α- and γ-tocopherol [46
], docosahexahenoic acid [39
], oleic acid [46
], polyphenols [82
], and dimethylfumarate [72
], MTSO also significantly reduced the oxidative stress revealed by DHE staining as well as the percentage of cells permeable to PI. This increased the values of PI positive cells either indicates an increase of dead cells and/or an increase of cells with damaged plasma membranes resulting from lipid peroxidation which could be the consequence of ROS overproduction [4
]. These cytoprotective effects of MTSO on the plasma membrane evocate those observed with argan oil which protects against the cytotoxic effects induced by 7KC [47
]. In addition, 7KC- and 24S-induced mitochondrial dysfunctions were attenuated by MTSO—the mitochondrial dysfunction revealed by the MTT test was attenuated. In addition, MTSO was able to prevent 7KC-induced LC3-I activation in LC3-II and the cleavage of caspase-3, which are the criteria for autophagy and apoptosis, respectively. Currently, the autophagic process associated with 7KC-induced cell death is considered rather beneficial [83
]. Since some compounds present in MTSO can normalize autophagy and attenuate apoptosis, which are two events involved in neurodegeneration, this is an important argument in favor of the potentially neuroprotective activity of MTSO.
So, it can be considered that MTSO is a mixture of compounds (fatty acids, polyphenols, tocopherols, and phytosterols) that can act synergistically. Polyphenols are mainly hydrophilic molecules present at very low levels in oil; their cytoprotective activity on 7KC-induced oxiapoptophagy can be excluded. On the other hand, fatty acids (especially oleic acid) and tocopherols (mainly α- and γ-tocopherol) are present at high concentrations, and it has already been proved that they prevent 7KC-induced oxiapoptophagy [34
]. It is, therefore, supposed that these compounds probably contribute to the cytoprotective effects of MTSO. Since several data are available on the signaling pathways activated by 7KC [85
] and on the cellular targets of tocopherols and oleic acid involved in the attenuation of 7KC-induced side effects [43
], it will be easy to determine whether this assumption is realistic.
It is noteworthy that in the presence of nigella seed oil, considered potentially toxic, major differences compared to MTSO are observed. Whereas MTSO is cytoprotective, nigella seed oil is not; it is cytotoxic and does not prevent 7KC- and 24S-induced side effects. In vivo, toxic effects of nigella seed oil have also been reported by Zaoui et al. [87
], who showed that certain blood parameters were modified, especially the platelet count. Other more recent in vivo studies by Zaghloul et al. [88
] confirmed the toxic effect of nigella seed oil on the renal cortex and, to a lesser degree, on hepatic cells. In addition, whereas the content of fatty acids is almost similar in MTSO and nigella seed oil, there are major differences in their sterol contents. It is suggested that, the cytotoxicity of nigella seed oil could be explained, at least in part, by the high content of cycloartenol and methylene cycloartenol which are not found in MTSO. Thymoquinone, which is a polyphenol known for its anti-inflammatory, antioxidant, and anti-proliferative properties, is also only found in MTSO, but its concentration is probably too low to induce side effects [89
Altogether our data support that our in vitro assay on 158N cells, which is rapid and easy to standardize, could be used as a screening test in order to distinguish and identify oils with different biological activities.
In addition, the present study provides new data that reinforce the interest in the use of MTSO for the prevention of neurodegenerative diseases. The chemical profile of MTSO was quite similar in the three areas of Tunisia studied (Bizerte, Zaghouan, and Sousse), and revealed the presence of fatty acids, polyphenols, phytosterols, and tocopherols capable of attenuating the side effects associated with neurodegeneration (oxidative stress, mitochondrial dysfunction, and apoptosis/autophagy). These chemical characteristics reinforce the interest in the potential of MTSO to prevent age-related diseases including neurodegenerative diseases, since several of its compounds are theoretically capable of crossing the blood–brain barrier and have been shown to have neurotrophic (cytoprotective + differentiating) properties on nerve cells, mainly oleic acid and polyphenols [90
]. In addition, MTSO has antioxidant properties and is also capable of counteracting 7KC- and 24S-induced oxiapoptophagy in 158N murine oligodendrocytes. Since 7KC and 24S can be increased in patients with neurodegenerative diseases, the cytoprotective effects of MTSO against the toxic effects of 7KC and 24S suggest that MTSO may have beneficial effects related to preventing or slowing down the development of 7KC- and 24S- associated neurodegenerative diseases. In addition, the results obtained on the cytoprotective effects of milk thistle seed oil on 158N cells encourage us to develop more elaborate cell models that take into account the selective passage of certain oil compounds through the blood–brain barrier. Models mimicking the blood–brain barrier by combining endothelial cells, pericytes, and nerve cells [92
] should provide a more accurate evaluation of the activity of MTSO on brain cells under normal and pathologic conditions induced by mechanical, physical and/or chemical agents.