Extraction, physicochemical characterization, lipid profile and cytoprotective activity of milk thistle seeds oil from Tunisia

La famille des Astéracées est économiquement très importante, car beaucoup de ses plantessont cultivées principalement pour leurs valeurs alimentaires dont la laitue (Lactuca), lachicorée (Cichorium) et le tournesol (Heliantus annuus) cultivé pour son huile. Une despropriétés typiques de cette famille, dont fait partie le Silybum marianum, est la richesse deses huiles en divers composés naturels bénéfiques pour la santé. Actuellement et pour lesdécennies à venir, les maladies liées à l’âge constituent un problème majeur de santépublique. Prévenir leur apparition ou s'opposer à leur évolution est un objectif majeur. Dansce contexte, les activités cytoprotectrices de l'huile de graines de Silybum marianumoriginaires de différentes régions de la Tunisie ont été étudiées sur le modèled’oligodendrocytes murins 158N en utilisant le 7-cétocholestérol (7KC) comme agentcytotoxique. Le 7KC a été utilisé car il est augmenté dans le plasma, le liquide céphalorachidienet/ou certains tissus (paroi vasculaire et intestinale, rétine, cristallin, cerveau) depatients atteints de maladies liées à l’âge (maladies cardiovasculaires, maladie d'Alzheimer,dégénérescence maculaire liée à l’âge, cataracte) ou de maladies inflammatoires évolutives del’intestin. Le 24S-hydroxycholestérol (24S-OHC) a aussi été utilisé car il est augmenté dans lecerveau aux stades précoces de la maladie d’Alzheimer. Afin de déterminer la composition del'huile de Silybum marianum (acides gras, tocophérols, polyphénols, phytostérols) diversestechniques physicochimiques et de chromatographie liquide et gazeuse ont été utilisées. Lespropriétés anti-oxydantes de l'huile de Silybum marianum ont été définies par les tests KRL,FRAP et DPPH et les propriétés cytoprotectrices par des techniques complémentaires demicroscopie, de cytométrie en flux et de biochimie. Les études sur la compositionphysicochimique des huiles des graines de Silybum marianum de différentes régions de laTunisie (Bizerte, Sousse, Zaghouan) ont montré que ces dernières sont riches en Vitamine E(α-tocophérol) et sont fortement anti-oxydantes. Les acides gras majeurs sont l'acidelinoléique (C18:2) (valeurs comprises entre 57,0% et 60,3%) et l'acide oléique (C18:1) (lesvaleurs se situent entre 15,5% et 22,4%). Trois acides phénoliques ont également étéidentifiés (vanillique, p-coumarique et silybine), avec une prédominance de l'acide vanillique.Sur les cellules 158N, ces huiles atténuent la cytotoxicité du 7KC et du 24S-OHC: perted'adhérence cellulaire, altération de la membrane plasmique, dysfonctionnementmitochondrial, surproduction d’espèces réactives de l’oxygène, induction de l'apoptose(condensation et / ou fragmentation nucléaire, activation de la caspase-3 et clivage PARP) etautophagie (activation de LC3-I en LC3-II). L'atténuation des effets cytotoxiques du 7KC etdu 24S-OHC observée avec les huiles de Silybum marianum est de l'ordre de celle observéeavec l’α-tocophérol utilisé comme témoin positif. La composition chimique et les résultatsobtenus cellules 158N sont en faveur d’effets bénéfiques de l’huile de Chardon-Marie sur lasanté humaine et la prévention de certaines maladies liées à l’âge.The family Asteraceae is economically very important because many of its plants are grownmainly for their food values including lettuce (Lactuca), chicory (Cichorium) and sunflower(Heliantus annuus) grown for its oil. One of the typical properties of this family, whichincludes Silybum marianum, is the richness of its oils in various natural compounds beneficialto health. Currently and for decades to come, age-related diseases are a major public healthproblem. Preventing their appearance or opposing their evolution is a major objective. In thiscontext, the cytoprotective activities of Silybum marianum seed oil originating from differentarea of Tunisia were studied on the 158N murine oligodendrocyte model using 7-ketocholesterol (7KC) and 24S-hydroxycholesterol (24S-OHC) as a cytotoxic agent. The 7KChas been used because it is increased in plasma, cerebrospinal fluid and / or tissues (vascularand intestinal wall, retina, lens, brain) of patients with age-related diseases (cardiovasculardiseases, Alzheimer's disease, age-related macular degeneration, cataract) or inflammatorybowel disease. The 24S-OHC was used because it is increased in the brain at the early stagesof Alzheimer’s disease. In order to determine the composition of Silybum marianum oil (fattyacids, tocopherols, polyphenols, phytosterols) various liquid and gas chromatographytechniques were used. The anti-oxidant properties of Silybum marianum oil were defined byKRL, FRAP and DPPH tests and cytoprotective properties by complementary microscopy,flow cytometry and biochemistry techniques. Studies on the physicochemical composition ofSilybum marianum seeds oils from different regions of Tunisia (Bizerte, Sousse, Zaghouan)have shown that the latter are rich in vitamin E (α-tocopherol) and are strongly antioxidant.The major fatty acids are linoleic acid (C18:2) (values between 57.0% and 60.3%) and oleicacid (C18:1) (values between 15.5% and 22.4%).. Three phenolic acids have also beenidentified (vanillic, p-coumaric and silybin), with a predominance of vanillic acid. On 158Ncells, these oils attenuate the cytotoxicity of 7KC and 24S-OHC: loss of cell adhesion,alteration of the plasma membrane, mitochondrial dysfunction, overproduction of reactiveoxygen species, induction of apoptosis (nuclear condensation and / or fragmentation). ,activation of caspase-3 and PARP cleavage) and autophagy (activation of LC3-I in LC3-II).The attenuation of cytotoxic effects of 7KC and 24S-OHC observed with Silybum marianumoils is of the order of that observed with α-tocopherol used as a positive control. The chemicalcomposition and results obtained on 158N cells are in favor of the beneficial effects of milkthistle oil on human health and on its ability to prevent some age-related diseases

Tunisian Milk Thistle: An Investigation of the Chemical Composition and the Characterization of Its Cold-Pressed Seed Oils

In this study, milk thistle seeds growing in different areas in Tunisia were cold pressed and the extracted oils were examined for their chemical and antioxidant properties. The major fatty acids were linoleic acid (C18:2) (57.0%, 60.0%, and 60.3% for the milk thistle seed oils native to Bizerte, Zaghouan and Sousse, respectively) and oleic acid (C18:1) (15.5%, 21.5%, and 22.4% for the milk thistle seed oils originating from Bizerte, Zaghouan and Sousse, respectively). High performance liquid chromatography (HPLC) analysis showed the richness of the milk thistle seed oils (MTSO) in α-tocopherol. The highest content was recorded for that of the region of Zaghouan (286.22 mg/kg). The total phenolic contents (TPC) of Zaghouan, Bizerte, and Sousse were 1.59, 8.12, and 4.73 Gallic Acid Equivalent (GAE) mg/g, respectively. Three phenolic acids were also identified (vanillic, p-coumaric, and silybine), with a predominance of the vanillic acid. The highest value was recorded for the Zaghouan milk thistle seed oil (83 mg/100 g). Differences in outcomes between regions may be due to climatic differences in areas. Zaghouan’s cold-pressed milk thistle seed oil had a better quality than those of Bizerte and Sousse, and can be considered as a valuable source for new multi-purpose products or by-products for industrial, cosmetic, and pharmaceutical utilization

Nigella and milk thistle seed oils: potential cytoprotective effects against 7β-hydroxycholesterol-induced toxicity on SH-SY5Y cells

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7β-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC.This study was supported by the Ministry of Higher Education and Scientific Research (MHESR), Tunisia. This work was also supported by the Spanish Ministry of Science and Innovation and FEDER Funds through grants SAF2017-83372-R (FJM) and through the “María de Maeztu” Program for Units of Excellence in R&D from Spain (award CEX2018-000792-M)

Induction of peroxisomal changes in oligodendrocytes treated with 7-ketocholesterol: Attenuation by α-tocopherol

This work was presented as an oral presentation at the 7th ENOR (European Network for Oxysterol Research) Symposium ‘Oxysterols and Sterol Derivatives in Health and Disease’, September 21–22, 2017, Université catholique de Louvain, Brussels, Belgium (https://www.oxysterols.net/).International audienceThe involvement of organelles in cell death is well established especially for endoplasmic reticulum, lysosomes and mitochondria. However, the role of the peroxisome is not well known, though peroxisomal dysfunction favors a rupture of redox equilibrium. To study the role of peroxisomes in cell death, 158 N murine oligodendrocytes were treated with 7-ketocholesterol (7 KC: 25-50 mu M, 24 h). The highest concentration is known to induce oxiapoptophagy (OXIdative stress + APOPTOsis + autoPHAGY), whereas the lowest concentration does not induce cell death. In those conditions (with 7 KC: 50 mu M) morphological, topographical and functional peroxisome alterations associated with modifications of the cytoplasmic distribution of mitochondria, with mitochondrial dysfunction (loss of transmembrane mitochondrial potential, decreased level of cardiolipins) and oxidative stress were observed: presence of peroxisomes with abnormal sizes and shapes similar to those observed in Zellweger fibroblasts, lower cellular level of ABCD3, used as a marker of peroxisomal mass, measured by flow cytometry, lower mRNA and protein levels (measured by RT-qPCR and western blotting) of ABCD1 and ABCD3 (two ATP-dependent peroxisomal transporters), and of ACOX1 and MFP2 enzymes, and lower mRNA level of DHAPAT, involved in peroxisomal beta-oxidation and plasmalogen synthesis, respectively, and increased levels of very long chain fatty acids (VLCFA: C24:0, C24:1, C26:0 and C26:1, quantified by gas chromatography coupled with mass spectrometry) metabolized by peroxisomal beta-oxidation. In the presence of 7 KC (25 mu M), slight mitochondrial dysfunction and oxidative stress were found, and no induction of apoptosis was detected; however, modifications of the cytoplasmic distribution of mitochondria and clusters of mitochondria were detected. The peroxisomal alterations observed with 7 KC (25 mu M) were similar to those with 7 KC (50 mu M). In addition, data obtained by transmission electron microcopy and immunofluorescence microscopy by dual staining with antibodies raised against p62, involved in autophagy, and ABCD3, support that 7 KC (25-50 mu M) induces pexophagy. 7 KC (25-50 mu M)-induced side effects were attenuated by alpha-tocopherol but not by alpha-tocotrienol, whereas the anti-oxidant properties of these molecules determined with the FRAP assay were in the same range. These data provide evidences that 7 KC, at concentrations inducing or not cell death, triggers morphological, topographical and functional peroxisomal alterations associated with minor or major mitochondrial changes

Profile of fatty acids, tocopherols, phytosterols and polyphenols in mediterranean oils (argan oils, olive oils, milk thistle seed oils and nigella seed oil) and evaluation of their antioxidant and cytoprotective activities

International audienceBackground: The effects of vegetable oils on human health depends on their components. Therefore, their profiles of lipid nutrients were determined. Objective: To establish and compare the fatty acid, tocopherol, phytosterol and polyphenol profiles of Mediterranean oils: cosmetic and dietary argan oils (AO; Morocco: Agadir, Berkane); olive oils (OO; Morocco, Spain, Tunisia); milk thistle seed oils (MTSO; Tunisia: Bizerte, Sousse, Zaghouane); nigella seed oil (NSO). Method: The biochemical profiles were determined by gas chromatography-flame ionization, high performance liquid chromatography and gas chromatography, coupled with mass spectrometry as required. The antioxidant and cytoprotective activities were evaluated with the KRL (Kit Radicaux Libres) and the fluorescein diacetate tests on nerve cells treated with 7-ketocholesterol (7KC). Results: The fatty acid profile revealed high linoleic acid (C18:2 n-6) content in AO, OO, MTSO and NSO. The highest levels of oleic acid (C18:1 n-9) were found in AO and OO. The tocopherol profile showed that Agadir AO contained the highest amount of α-tocopherol, also present at high level in MTSO and Tunisian OO; Berkane AO was rich in γ-tocopherol. The phytosterol profile indicated that β-sitosterol was predominant in the oils, except AO; spinasterol was only present in AO. Polyphenol profiles underlined that OO was the richest in polyphenols; hydroxytyrosol was only in OO; few polyphenols were detected in AO. The oils studied have antioxidant activities, and all of them, except NSO, prevented 7KC-induced cell death. The antioxidant characteristics of AO were positively correlated with procatechic acid and compestanol levels. Conclusion: Based on their biochemical profiles, antioxidant and cytoprotective characteristics, AO, OO, and MTSO are potentially beneficial to human health

Comparison of the effects of major fatty acids present in the Mediterranean diet (oleic acid, docosahexaenoic acid) and in hydrogenated oils (elaidic acid) on 7-ketocholesterol-induced oxiapoptophagy in microglial BV-2 cells

International audienceIncreased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, reactive oxygen species overproduction and lipid peroxidation, increased plasma membrane permeability and fluidity, nuclei condensation and/or fragmentation and caspase-3 activation, which are apoptotic characteristics, and an increased LC3-II/LC3-I ratio, which is a criterion of autophagy. 7KC is therefore a potent inducer of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY) on BV-2 cells. OA and EA, but not DHA, also favor the accumulation of lipid droplets revealed with Masson's trichrome, Oil Red O, and Nile Red staining. The cytotoxicity of 7KC was strongly attenuated by OA and DHA. Protective effects were also observed with EA. However, 7KC-induced caspase-3 activation was less attenuated with EA. Different effects of OA and EA on autophagy were also observed. In addition, EA (but not OA) increased plasma membrane fluidity, and only OA (but not EA) was able to prevent the 7KC-induced increase in plasma membrane fluidity. Thus, in BV-2 microglial cells, the principal fatty acids of the Mediterranean diet (OA, DHA) were able to attenuate the major toxic effects of 7KC, thus reinforcing the interest of natural compounds present in the Mediterranean diet to prevent the development of neurodegenerative diseases

Protective effects of milk thistle (Sylibum marianum) seed oil and α-tocopherol against 7β-hydroxycholesterol-induced peroxisomal alterations in murine C2C12 myoblasts: Nutritional insights associated with the concept of pexotherapy

International audiencePeroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7β-hydroxycholesterol (7β-OHC; 50 μM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 μg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7β-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7β-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7β-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7β-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal β-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal β-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy)

Argan Oil-Mediated Attenuation of Organelle Dysfunction, Oxidative Stress and Cell Death Induced by 7-Ketocholesterol in Murine Oligodendrocytes 158N

Argan oil is widely used in Morocco in traditional medicine. Its ability to treat cardiovascular diseases is well-established. However, nothing is known about its effects on neurodegenerative diseases, which are often associated with increased oxidative stress leading to lipid peroxidation and the formation of 7-ketocholesterol (7KC) resulting from cholesterol auto-oxidation. As 7KC induces oxidative stress, inflammation and cell death, it is important to identify compounds able to impair its harmful effects. These compounds may be either natural or synthetic molecules or mixtures of molecules such as oils. In this context: (i) the lipid profiles of dietary argan oils from Berkane and Agadir (Morocco) in fatty acids, phytosterols, tocopherols and polyphenols were determined by different chromatographic techniques; and (ii) their anti-oxidant and cytoprotective effects in 158N murine oligodendrocytes cultured with 7KC (25–50 µM; 24 h) without and with argan oil (0.1% v/v) or α-tocopherol (400 µM, positive control) were evaluated with complementary techniques of cellular and molecular biology. Among the unsaturated fatty acids present in argan oils, oleate (C18:1 n-9) and linoleate (C18:1 n-6) were the most abundant; the highest quantities of saturated fatty acids were palmitate (C16:0) and stearate (C18:0). Several phytosterols were found, mainly schottenol and spinasterol (specific to argan oil), cycloartenol, β-amyrin and citrostadienol. α- and γ-tocopherols were also present. Tyrosol and protocatechic acid were the only polyphenols detected. Argan and extra virgin olive oils have many compounds in common, principally oleate and linoleate, and tocopherols. Kit Radicaux Libres (KRL) and ferric reducing antioxidant power (FRAP) tests showed that argan and extra virgin olive oils have anti-oxidant properties. Argan oils were able to attenuate the cytotoxic effects of 7KC on 158N cells: loss of cell adhesion, cell growth inhibition, increased plasma membrane permeability, mitochondrial, peroxisomal and lysosomal dysfunction, and the induction of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY). Altogether, our data obtained in 158N oligodendrocytes provide evidence that argan oil is able to counteract the toxic effects of 7KC on nerve cells, thus suggesting that some of its compounds could prevent or mitigate neurodegenerative diseases to the extent that they are able to cross the blood‐brain barrier