Diet and its components play major roles in the onset and development of several degenerative diseases such as atherosclerosis, CVD, cancer and neurodegeneration(Reference McNaughton, Mishra and Brunner1). In this respect, adherence to the Mediterranean diet is associated with lower incidence of CVD and cancer(Reference Martinez-Gonzalez, Bes-Rastrollo and Serra-Majem2, Reference Bedard, Goulet and Riverin3). It must be emphasised that the term ‘Mediterranean diet’ encompasses several diverse dietary profiles typical of the Mediterranean basin(Reference Bogani and Visioli4, Reference Bere and Brug5). Indeed, some differences, e.g. with respect to alcohol consumption or protein intake, exist among the various Mediterranean countries(Reference Bere and Brug5). Among the various components of the Mediterranean diet, much attention is being paid to olive oil, because its consumption is associated with favourable cardiovascular outcomes(Reference Visioli and Bernardini6). However, some countries, namely those in the Maghreb area, also use argan oil for culinary and cosmetic applications(Reference Charrouf and Guillaume7). Indeed, accumulating evidence suggests that argan oil might be endowed with potential healthful properties, mostly in the areas of CVD(Reference Drissi, Girona and Cherki8) and prostate cancer(Reference Bennani, Drissi and Giton9). As an example, several recent studies reported hypolipidaemic, hypocholesterolaemic and antihypertensive effects of argan oil in the rat(Reference Charrouf and Guillaume7, Reference Berrougui, Ettaib and Herrera10). Argan oil is obtained from the fruit of Argania spinosa (Sapotaceae), an endemic tree which mostly grows in South-western Morocco. Argan trees also grow in Algeria, namely in the Tindouf countryside. While technological advancement, e.g. Soxlet's extraction, is changing argan oil production, most of its making still follows traditional procedures, i.e. hand-grounding and cold-pressing of the kernels, yielding yellowish, nutty-flavoured products(Reference Charrouf and Guillaume7). The fatty acid profile of argan oil consists of approximately 45 % MUFA, approximately 35 % PUFA and approximately 20 % SFA. Moreover, virgin argan oil contains minor, bioactive components such as phenolic compounds, phytosterols and tocopherols(Reference Khallouki, Younos and Soulimani11, Reference Cherki, Berrougui and Drissi12).
Despite suggestive in vitro and animal evidence, the potential cardioprotective properties of argan oil have been the subject of very limited studies in humans(Reference Drissi, Girona and Cherki8, Reference Cherki, Derouiche and Drissi13). Moreover, the in vivo effects of Algerian argan oil have never been investigated. Therefore, we sought to comprehensively determine the effects of argan oil supplementation on plasma lipid profile and antioxidant status of a group of healthy Algerian subjects, compared with matched controls.
Experimental methods
Materials and methods
Argan fruits were harvested in the Tindouf area of South-western Algeria. The argan oil used in the present work was produced from freshly picked seeds of a single harvest, using traditional hand-methods. The oil was kept at 4°C in a brown glass bottle until the beginning of the trial.
The fatty acid composition of the oil was determined by gas-phase chromatography (Applied Sciences Labs, State College, PA, USA). Fatty acid standards were from Nu-Check-Prep (Elysian, MN, USA). Vitamin E (α-tocopherol) was measured by HPLC according to Zaman et al. (Reference Zaman, Fielden and Frost14). Polyphenols were extracted from the oil and quantified using the method of Pirisi et al. (Reference Pirisi, Cabras and Cao15). The composition of the oil at study is given in Table 1. More than 80 % of total fatty acids was composed of oleic and linoleic acids (45·01 and 35·39 %, respectively). Linolenic acid accounted for only 0·2 % of total fatty acids. SFA (mostly palmitic and stearic acids) accounted for 17·7 % of total fatty acids. This oil was devoid of erucic acid. Vitamin E (α-tocopherol) concentration was quite low, i.e. 56·34 mg/kg. Polyphenols were also scarcely present, i.e. 52·36 mg/kg.
Composition of the argan oil adopted in this study
Subjects
The present study conforms to the Declaration of Helsinki (Ethical Principles for Medical Research Involving Human Subjects) and was approved by the local ethics committee. A total of forty healthy subjects aged 25–45 years with normal BMI and blood pressure were recruited from within the Abu Bakr Belkaid University (Tlemcen, Algeria) and written or oral informed consent to the study was taken from them. All participants were free of metabolic diseases such as hypercholesterolaemia, hypertriacylglycerolaemia, diabetes and hypertension, and were non-smokers and medication-free. We monitored the volunteers' lifestyle, e.g. physical activity, working hours and sleep duration, which did not markedly change throughout this study. A total of twenty subjects consumed 15 g/d of argan oil – with toasted bread – for breakfast, during 4 weeks (intervention group), whereas twenty matched controls followed their habitual diet, but did not consume argan oil. We found that provision of argan oil translated into lower consumption of habitual fat (oils and, to a lower extent, butter). It is noteworthy that, unexpectedly, consumption of olive oil in Algeria is quite low (approximately 1 kg/year), whereas milk consumption is higher than in neighbouring Maghreb countries(Reference Kallithraka, el-Jazouli and Zeghichi16, Reference Zeghichi-Hamri and Kallithraka17).
We chose the dose of 15 g/d because (a) it was well tolerated by our volunteers and approximates habitual consumption and (b) Drissi et al. (Reference Drissi, Girona and Cherki8) previously reported on the healthful effects of this daily dose.
Both groups had similar anthropometric characteristics (Table 2). All subjects filled in a food questionnaire, in which they noted the quality and quantity of food consumed during the day before blood sampling, including their argan oil intake (which was nil in controls). The registered values were converted into energy and were estimated according to the Ciqual standard table of food composition(18). All participants had similar physical activity and lifestyle.
(Mean values and standard deviations)
SBP, systolic blood pressure; DBP, diastolic blood pressure.
Blood analyses
At days 0 (T 0), 15 (T 15) and 30 (T 30), venous blood was drawn into evacuated tubes, some of which contained disodium EDTA as the anticoagulant; other tubes did not contain anticoagulants to allow for the preparation of serum. Both serum and plasma were separated by centrifugation at 2100 g for 20 min at 4°C, aliquoted, and stored at − 20°C. Erythrocytes were collected and washed three times in isotonic saline; then they were haemolysed by the addition of cold distilled water (1/4, vol/vol). Cellular debris was removed by centrifugation.
Lipoprotein and lipid determination
Plasma lipoproteins (LDL, d < 1·063; HDL, d < 1·21 g/ml) were separated by sequential ultracentrifugation. Serum total cholesterol (TC) and TAG were measured using enzymatic kits (Quimica Clinica Aplicada S.A., Amposta, Spain). HDL-cholesterol and LDL-cholesterol concentrations were also measured by enzymatic kits.
Determination of hydroperoxides
Both plasma and erythrocyte levels of hydroperoxides (lipoperoxides, LOOH) were measured by the ferrous ion oxidation-xylenol orange assay – using the specific LOOH reducer triphenylphosphine – according to the method of Nourooz-Zadeh et al. (Reference Nourooz-Zadeh, Tajaddini-Sarmadi and Ling19).
Determination of thiobarbituric acid-reacting substances
Thiobarbituric acid-reacting substances (TBARS) in plasma and erythrocytes were measured according to the method of Nourooz-Zadeh et al. (Reference Nourooz-Zadeh, Tajaddini-Sarmadi and Ling19).
Determination of carbonyl proteins
Carbonyl proteins were measured in plasma and erythrocytes by the 2,4-dinitrophenylhydrazine reaction described by Levine et al. (Reference Levine, Garland and Oliver20).
Conjugated diene formation
The in vitro oxidisability of plasma lipoproteins induced by metals, i.e. copper sulphate, was analysed by monitoring, over time, the formation of conjugated dienes, as described by Esterbauer et al. (Reference Esterbauer, Striegl and Puhl21).
Determination of plasma levels of vitamins A, E and C
Vitamins A (retinol) and E (α-tocopherol) were determined in the plasma of all volunteers by HPLC coupled with UV detection at 292 nm for vitamin E and 325 nm for vitamin A, according to Zaman et al. (Reference Zaman, Fielden and Frost14). Vitamin C was measured in plasma by the method of Jagota & Dani(Reference Jagota and Dani22).
Determination of catalase activity
Catalase (CAT; EC 1.11.1.6) activity was measured by spectrophotometric analysis of the rate of H2O2 decomposition at 240 nm, according to the method of Aebi(Reference Aebi and Bergmeyer23).
Oxygen radical absorbance capacity
The total antioxidant ability of plasma (oxygen radical absorbance capacity, ORAC) was estimated by the capacity of erythrocytes to resist free-radical-induced haemolysis, according to the method of Blache & Prost(Reference Blache and Post24).
Statistical analysis
Results are expressed as means and standard deviations. The Student's t test was used to compare data from the intervention group with those from controls. Statistical analysis was performed using Statistica (version 4.1, Statsoft, Paris, France). A P value less than 0·05 was considered as statistically significant.
Results
Dietary profile
Neither total daily energy intake nor consumption of proteins, carbohydrates and lipids significantly differed between the two groups during the experiment (Table 3). Consumption of SFA and MUFA was also similar in both groups; however, argan oil intake resulted in a higher overall consumption of PUFA (Table 3).
Dietary intakes at day 30
* Mean values were significantly different (P < 0·05).
Circulating lipids
We recorded a significant improvement in the plasma lipid profile of subjects who were given argan oil as compared to controls, whose values did not significantly change during the study (data not shown). In particular, TAG decreased by 20·97 %, TC by 14·63 % and LDL-cholesterol by 16·05 % after 30 d of treatment (Table 4). We also recorded a concomitant non-significant increase in HDL-cholesterol.
(Mean values and standard deviations)
T 0, day 0; T 15, day 15; T 30, day 30; TC, total cholesterol; HDL-C, HDL-cholesterol; LDL-C, LDL-cholesterol.
Mean values were significantly different from those of controls: *P < 0·05, **P < 0·001.
† The plasma lipid profile of control subjects did not change significantly throughout the study: we present the average value of T 0+T 15+T 30.
Circulating and cellular markers of oxidation and antioxidant status
Circulating and cellular markers of lipid (TBARS and LOOH) and protein (carbonyls) oxidation are shown in Table 5. Argan oil significantly decreased the former (at T 30), but not the latter. In addition, susceptibility of LDL to copper-induced oxidation was decreased in argan oil-treated subjects, as indicated by the significantly increased lag phase and decrease of conjugated diene production (Table 6).
(Mean values and standard deviations)
T 0, day 0; T 15, day 15; T 30, day 30; TBARS, thiobarbituric acid-reacting substances; pl, plasma; LOOH, lipoperoxides; PC, protein carbonyls.
Mean values were significanty different from those of controls: *P < 0·05, **P < 0·01.
† The biomarkers of control subjects did not change significantly throughout the study: we present the average value of T 0+T 15+T 30.
(Mean values and standard deviations)
T 0, day 0; T 15, day 15; T 30, day 30; LP, lag phase; MR, maximal rate; IDP, initial conjugated diene production; MDP, maximum conjugated diene production.
* Mean values were significantly different from those of controls (P < 0·05).
† The susceptibility of LDL to oxidation of control subjects did not change significantly throughout the study: we present the average value of T 0+T 15+T 30.
Plasma vitamin E concentrations were significantly increased (+18 % at T 15, P < 0·05 and +43 % at T 30, P < 0·001 when compared with controls; Table 7) by argan oil ingestion throughout the study. Conversely, vitamins A and C did not change significantly in either group. CAT activity also increased significantly in argan oil-treated subjects, whereas plasma ORAC remained unmodified.
(Mean values and standard deviations)
T 0, day 0; T 15, day 15; T 30, day 30; ORAC, oxygen radical absorbance capacity; AU, arbitrary; CAT, catalase; pl, plasma.
Mean values were significantly different from those of controls: *P < 0·05, **P < 0·01.
Discussion
We performed the first trial of Algerian argan oil in human subjects and evaluated a wide array of surrogate markers of CVD. Our present study results show that daily consumption of feasible amounts of argan oil positively modulates such markers. Argan oil consumption is increasing in Europe and high-quality virgin argan oil can also be currently purchased in Japan and the USA, where it is mostly sold for its purported cosmetic properties. Thus far, due to its elevated price, the dietary use of argan oil is mostly limited to the areas where it is produced (for the most part Morocco, although West Algeria also contributes). As such, it contributes to the diet of selected population groups and is an integral part of the Maghrebian version of the Mediterranean diet.
Our results are in agreement with those of Drissi et al. (Reference Drissi, Girona and Cherki8) and of Cherki et al. (Reference Cherki, Derouiche and Drissi13) and fit with cumulated animal evidence of the anti-atherogenic potential of argan oil(Reference Charrouf and Guillaume7). In particular, argan oil consumption – in feasible amounts – decreased total and LDL-cholesterol and TAG. Of note, our subjects had low mean cholesterolaemia, which reflects the average values found in countryside Algeria. Other cardiovascular parameters such as blood pressure were not modified by argan oil consumption, in contrast with the findings of Berrougui et al. (Reference Berrougui, Alvarez and Perez-Guerrero25) who reported hypotensive effects in normotensive Wistar rats. This discrepancy might be due to species specificity, dose or treatment duration.
The exact mechanisms of action by which argan oil exerts its lipid-modulating and potentially anti-atherogenic actions remain elusive. However, we speculate that the high proportion of PUFA and MUFA in the argan oil we administered – and reflected in the overall dietary intake (Table 3) – played marked roles, as suggested by the available literature(Reference Ramsden, Hibbeln and Majchrzak26, Reference Calder27). Other contributors to the hypolipidaemic effects of argan oil are – probably – sterols and saponins, which we did not measure in the present investigation due to technical limitations, but which have been previously investigated by Khallouki et al. (Reference Khallouki, Younos and Soulimani11) and Berrougui et al. (Reference Berrougui, Cloutier and Isabelle28). Both classes of molecules interfere with intestinal cholesterol absorption and are currently exploited as functional food components(Reference Marangoni and Poli29).
We also evaluated circulating markers of oxidation and report that argan oil consumers had lower concentrations of LOOH and TBARS, but not of oxidatively-modified proteins. Total antioxidant capacity (evaluated as ORAC) also did not change. Finally, we recorded increased intracellular CAT activity. This finding is, indeed, counterintuitive (there should be no reason to activate an antioxidant enzyme if the antioxidant profile is augmented). However, several studies with antioxidants, e. g. polyphenols, reported similar increases in the activity of antioxidant enzymes, namely superoxide dismutase and CAT (Reference Hassimotto, Pinto and Lajolo30). As far as argan oil is concerned, our results agreed with those of Benajiba et al. (Reference Benajiba, Morel and De Leiris31), which showed that the activities of cytosolic CAT were significantly higher in Wistar rats treated with argan oil in comparison with the untreated rats. We can conceivably attribute the observed antioxidant actions (including increased LDL resistance to oxidation, which confirms previous findings of Cherki et al. (Reference Cherki, Derouiche and Drissi13)) to the provision of vitamin E (which increased in consumers' plasma, Table 7) by argan oil. While Algerian argan oil appears to contain less vitamin E than, for example, the average Moroccan argan oil or extra-virgin olive oil, the contribution of tocopherols to the overall intake is, apparently, sufficient to elicit antioxidant effects. Other components such as polyphenols, namely ferulic acid (Reference Zougagh, Salghi and Dhair32), and sterols might have played a role. The precise nature of the cumulative positive effects on antioxidant profile, however, is still elusive and requires ad hoc investigations.
Our present study has several limitations – most of which are due to technical constraints – which we should acknowledge. As mentioned, we could not analyse the argan oil's content of sterols and saponins and, thus, we rely on the data of Khallouki et al. (Reference Khallouki, Younos and Soulimani11) and Berrougui et al. (Reference Berrougui, Cloutier and Isabelle28). We also could not evaluate the modifications of plasma and lipoprotein lipid profile brought about by consumption of argan oil. Hence, we rely on food composition tables to estimate intakes. Finally, we assessed an array of lipid peroxidation indices, due to the current lack of robust biomarkers(Reference Spickett, Wiswedel and Siems33); however, each of them has shortcomings(Reference Halliwell34).
In conclusion, we showed that argan oil is able to positively modulate some surrogate markers of CVD, through mechanisms which warrant further investigation. Moreover, based on these results, argan oil could prove to be an effective livelihood opportunity to diversify the options of small rural Berber producers and enhance their incomes.
Acknowledgements
We thank all the volunteers for their cooperation. We also thank the UPRES Laboratory of Lipids and Nutrition, Faculty of Sciences Gabriel, Burgundy University (Dijon, France). All authors read and approved the manuscript. S. S., D. K., N. B., N. S. and A. N. recruited the volunteers, analysed the FFQ and performed the biochemical analyses. M. B., F. C. and F. V. supervised the study and wrote the paper. All authors declare that there are no conflicts of interest. The present research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
